Antibodies to human gdf8

ABSTRACT

The present invention provides isolated human or humanized antibodies or antigen-binding fragments thereof which specifically bind to Growth and Differentiation Factor-8 (GDF8) and block GDF8 activity. The antibodies and antibody fragments of the present invention may be used in therapeutic methods for treating conditions or disorders which are ameliorated or improved by inhibition of GDF8.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/115,170, filed May 25, 2011, which claims the benefit under 35 U.S.C.§119(e) of U.S. provisional application No. 61/348,559, filed on May 26,2010; and 61/372,882, filed on Aug. 12, 2010, the disclosures of each ofwhich are herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for growth and differentiationfactor-8 (GDF8).

BACKGROUND

Growth and Differentiation Factor-8 (GDF8), also known as myostatin, isa member of the TGF-β superfamily of growth factors. GDF8 is a negativeregulator of skeletal muscle mass, highly expressed in the developingand adult skeletal muscle.

GDF8 is highly conserved across species, and the amino acid sequences ofmurine and human GDF8 are identical (human GDF8 nucleic acid sequenceand amino acid sequence shown in SEQ ID NO:338-339) (McPherron et al.1977 Nature 387:83-90).

A number of human diseases are associated with loss or impairment ofmuscle tissue, for example, muscular dystrophy, muscle atrophy, musclewasting syndrome, sarcopenia and cachexia, and inhibitors of GDF8 areapplicable to treating these diseases or disorders.

Antibodies to GDF8 and therapeutic methods are disclosed in, e.g., U.S.Pat. No. 6,096,506, U.S. Pat. No. 7,320,789, U.S. Pat. No. 7,807,159, WO2007/047112, WO 2005/094446, US 2007/0087000, U.S. Pat. No. 7,261,893,and WO 2010/070094.

BRIEF SUMMARY OF THE INVENTION

The present invention provides human or humanized antibodies andantigen-binding fragments of human or humanized antibodies thatspecifically bind human growth and differentiation factor 8 (GDF8).These antibodies are characterized by binding to GDF8 with high affinityand by the ability to neutralize GDF8 activity. The antibodies can befull-length (for example, an IgG1 or IgG4 antibody) or may comprise onlyan antigen-binding portion (for example, a Fab, F(ab′)₂ or scFvfragment), and may be modified to affect functionality, e.g., toeliminate residual effector functions (Reddy et al. (2000) J. Immunol.164:1925-1933).

In one embodiment, the antibody of the invention comprises a heavy chainvariable region (HCVR) amino acid sequence selected from the groupconsisting of SEQ ID NO:2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162,178, 194, 210, 226, 242, 258, 274, 290, 306, 360, and 376, or asubstantially identical sequence thereof.

In one embodiment, the antibody of the invention comprises a light chainvariable region (LCVR) amino acid sequence selected from the groupconsisting of SEQ ID NO:10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170,186, 202, 218, 234, 250, 266, 282, 298, 314, 322, 368, and 384 or asubstantially identical sequence thereof.

In one embodiment, the antibody of the invention comprises a HCVR aminoacid sequence and a LCVR amino acid sequence, wherein the HCVR/LCVR pairsequences are selected from the group consisting of SEQ ID NO:2/10,18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154,162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282,290/298, 306/314, 114/322, 360/368, and 376/384.

The present invention also features a human or humanized antibody orantigen-binding fragment of an antibody comprising a heavy chaincomplementarity determining region 3 (HCDR3) amino acid sequence and alight chain CDR3 amino acid sequence (LCDR3), wherein the HCDR3 aminoacid sequence is selected from the group consisting of SEQ ID NO:8, 24,40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264,280, 296, 312, 366, and 382, or a substantially identical sequencethereof, and the LCDR3 amino acid sequence is selected from the groupconsisting of SEQ ID NO:16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176,192, 208, 224, 240, 256, 272, 288, 304, 320, 328, 374, and 390, or asubstantially identical sequence thereof. In another embodiment, theantibody or fragment thereof comprises an HCDR3/LCDR3 amino acidsequence pair selected from the group consisting of SEQ ID NO:8/16,24/32, 40/48, 56/64, 72/80, 88/96, 104/112, 120/128, 136/144, 152/160,168/176, 184/192, 200/208, 216/224, 232/240, 248/256, 264/272, 280/288,296/304, 312/320, 120/328, 366/374, and 382/390.

In a related embodiment, the antibody or fragment thereof furthercomprises heavy chain CDR1 (HCDR1) and CDR2 (HCDR2) amino acid sequencesand light chain CDR1 (LCDR1) and CDR2 (LCDR2) amino acid sequences,wherein the HCDR1 amino acid sequence is selected from the groupconsisting of SEQ ID NO:4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164,180, 196, 212, 228, 244, 260, 276, 292, 308, 362, and 378, or asubstantially identical sequence thereof; the HCDR2 amino acid sequenceis selected from the group consisting of SEQ ID NO:6, 22, 38, 54, 70,86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294,310, 364, and 380, or a substantially identical sequence thereof; theLCDR1 amino acid sequence is selected from the group consisting of SEQID NO:12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220,236, 252, 268, 284, 300, 316, 324, 370, and 386 or a substantiallyidentical sequence thereof; and the LCDR2 amino acid sequence isselected from the group consisting of SEQ ID NO:14, 30, 46, 62, 78, 94,110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318,326, 372, and 388 or a substantially identical sequence thereof. Inanother embodiment, the HCDR1, HCDR2 and HCDR3 are selected from thegroup consisting of SEQ ID NO:36/38/40, 116/118/120, 228/230/232,362/364/366, and 378/380/382; and LCDR1, LCDR2 and LCDR3 are selectedfrom the group consisting of SEQ ID NO:44/46/48, 124/126/128,236/238/240, 370/372/374, and 386/388/390. In yet another embodiment,the heavy and light chain CDRs are selected from the group consisting ofSEQ ID NO: 36/38/40/44/46/48 (e.g. 21-E5), 116/118/120/124/126/128 (e.g.8D12), 228/230/232/236/238/240 (e.g. 1A2), 362/364/366/370/372/374 (e.g.H4H1657N2), and 378/380/382/386/388/390 (e.g. H4H1669P).

In a related embodiment, the invention includes an antibody orantigen-binding fragment of an antibody which specifically binds GDF8,wherein the antibody or fragment comprises the heavy and light chain CDRdomains contained within heavy and light chain variable domain sequencesselected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298,306/314, 114/322, 360/368, and 376/384. Methods and techniques foridentifying CDRs within HCVR and LCVR amino acid sequences are wellknown in the art and can be used to identify CDRs within the specifiedHCVR and/or LCVR amino acid sequences disclosed herein. Exemplaryconventions that can be used to identify the boundaries of CDRs include,e.g., the Kabat definition, the Chothia definition, and the AbMdefinition. In general terms, the Kabat definition is based on sequencevariability, the Chothia definition is based on the location of thestructural loop regions, and the AbM definition is a compromise betweenthe Kabat and Chothia approaches. See, e.g., Kabat, “Sequences ofProteins of Immunological Interest,” National Institutes of Health,Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948(1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272(1989). Public databases are also available for identifying CDRsequences within an antibody.

The present invention also provides nucleic acid molecules encoding theantibodies or antigen-binding fragments of the invention. Recombinantexpression vectors carrying the antibody-encoding nucleic acids of theinvention, and host cells into which such vectors have been introduced,are also encompassed by the invention, as are methods of making theantibodies of the invention by culturing the host cells of theinvention.

In one embodiment, the antibody of the invention comprises a HCVRencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO:1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209,225, 241, 257, 273, 289, 305, 359, and 375, or a substantially similarsequence having at least 95% homology thereof.

In one embodiment, the antibody of the invention comprises a LCVRencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO:9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217,233, 249, 265, 281, 297, 313, 321, 367, and 383 or a substantiallysimilar sequence having at least 95% homology thereof.

In one embodiment, the antibody of the invention comprises a HCVR aminoacid sequence and a LCVR amino acid sequence, wherein the HCV/LCVR pairsequences are encoded by a nucleic acid molecule pair selected from thegroup consisting of SEQ ID NO: 1/9, 17/25, 33/41, 49/57, 65/73, 81/89,97/105, 113/121, 129/137, 145/153, 161/169, 177/185, 193/201, 209/217,225/233, 241/249, 257/265, 273/281, 289/297, 305/313, 113/321, 359/367,and 375/383.

The present invention also features a human or humanized antibody orantibody fragment comprising a HCDR3 encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:7, 23, 39, 55, 71, 87,103, 119, 135, 151, 167, 183, 199, 215, 231, 247, 263, 279, 295, 311,365, and 381, or a substantially similar sequence having at least 95%homology thereof and a LCDR3 encoded by a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:15, 31, 47, 63, 79, 95, 111, 127,143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, 319, 327, 373,and 389, or a substantially similar sequence having at least 95%homology thereof. In one embodiment, the HCDR3/LCDR3 set is encoded by anucleotide sequence pair selected from the group consisting of SEQ IDNO:7/15, 23/31, 39/47, 55/63, 71/79, 87/95, 103/111, 119/127, 135/143,151/159, 167/175, 183/191, 199/207, 215/223, 231/239, 247/255, 263/271,279/287, 295/303, 311/319, 119/327, 365/373, and 381/389.

In a related embodiment, the antibody or antibody fragment furthercomprises a HCDR1 and HCDR2, and a LCDR1 and LCDR2, wherein the HCDR1 isencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO:3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211,227, 243, 259, 275, 291, 307, 361, and 377, or a substantially similarsequence having at least 95% homology thereof, the HCDR2 is encoded by anucleotide sequence selected from the group consisting of SEQ ID NO:5,21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245,261, 277, 293, 309, 363, and 379, or a substantially similar sequencehaving at least 95% homology thereof, the LCDR1 is encoded by anucleotide sequence selected from the group consisting of SEQ ID NO:11,27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251,267, 283, 299, 315, 323, 369, and 385 or a substantially similarsequence having at least 95% homology thereof, and the LCDR2 encoded bya nucleotide sequence selected from the group consisting of SEQ IDNO:13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237,253, 269, 285, 301, 317, 325, 371, and 387, or a substantially similarsequence having at least 95% homology thereof. In one embodiment, theantibody or antibody fragment comprises heavy and light chain CDRsencoded by a nucleic acid sequence set of SEQ ID NO:35/37/39/43/45/47,115/117/119/123/125/127, 227/229/231/235/237/239,361/363/365/369/371/373, or 377/379/381/385/387/389.

The present invention also features an isolated antibody or antibodyfragment that specifically binds GDF8, comprising heavy and light chainCDRs selected from the group consisting of (a) a HCDR1 comprising anamino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ IDNO:329), wherein X¹ is Gly; X² is Phe; X³ is Thr; X⁴ is Phe; X⁵ is Ser;X⁶ is Ala or Ser; X⁷ is Phe or Tyr; X⁸ is Gly or Ala; (b) a HCDR2comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸(SEQ ID NO:330), wherein X¹ is Ile; X² Gly or Ser; X³ is Tyr or Gly; X⁴Ser or Asp; X⁵ is Gly; X⁶ is Gly; X⁷ is Ser or Asn; and X⁸ is Ala orGlu; (c) a HCDR3 comprising an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴ (SEQ ID NO:331), whereinX¹ is Ser or Ala; X² is Thr or Lys; X³ is Asp or Ile; X⁴ is Gly or Ser;X⁵ is Ala or His; X⁶ is Trp or Tyr; X⁷ is Lys or Asp; X⁸ is Met or Ile;X⁹ is Ser or Leu; X¹⁰ is Gly or Ser; X¹¹ is Leu or Gly; X¹² is Asp orMet; X¹³ is Val or Asp; X¹⁴ is Val or absent; (d) a LCDR1 comprising anamino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶ (SEQ ID NO:332),wherein X¹ is Gln; X² is Asp or Gly; X³ is Ile; X⁴ is Ser; X⁵ is Asp orAsn; and X⁶ is Tyr or Trp; (e) a LCDR2 comprising an amino acid sequenceof the formula X¹-X²-X³ (SEQ ID NO:333), wherein X¹ is Thr or Ala; X² isThr or Ala; and X³ is Ser; and (f) a LCDR3 region comprising an aminoacid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:334),wherein X¹ is Gln; X² is Lys or Gln; X³ is Ala or Tyr; X⁴ is Asp or Asn;X⁵ is Ser; X⁶ is Ala or Phe; X⁷ is Pro; X⁸ is Leu; and X⁹ is Thr.

The methodology for deriving the aforementioned consensus sequences (SEQID NOs: 329-334) is illustrated in FIGS. 4A and 4B.

The present invention also features a fully human or humanized antibodyor antibody fragment which binds GDF8 with an affinity (expressed as adissociation constant, “K_(D)”) of about 1 nM or less, as measured bysurface plasmon resonance assay (for example, BIACORE™). In certainembodiments, the antibody of the invention exhibits a K_(D) of about 700pM or less; about 500 pM or less; about 320 pM or less; about 160 pM orless; about 100 pM or less; about 50 pM or less; about 10 pM or less; orabout 5 pM or less.

In one embodiment, the invention provides a fully human or humanizedmonoclonal antibody (mAb) which specifically binds and inhibits humanGDF8 and exhibits an IC₅₀ of less than or equal to about 10 nM; about 5nM or less; about 3 nM or less; about 2 nM or less; about 1 nM or less;about 500 pM or less; or about 200 pM or less, as measured by GDF8inducible luciferase assay. As shown in the experimental section below,some of the anti-GDF8 antibodies of the invention block the activity ofclosely related proteins, such as GDF11, with a much higher IC₅₀ thanGDF8 in a luciferase bioassay. In one embodiment, the invention providesan antibody or antigen-binding fragment of an antibody that exhibits atleast about 10-fold, at least about 50-fold, at least about 100-fold, atleast about 200-fold, at least about 500-fold, at least about 1000-fold,or at least about 1500-fold higher IC₅₀ for blocking GDF11 activityrelative to GDF8.

The invention encompasses anti-GDF8 antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof a galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

The invention includes anti-GDF8 antibodies which bind specific epitopesof GDF8 and are capable of blocking the biological activity of GDF8. Ina first embodiment, the antibody of the invention binds an epitope ofthe mature GDF8 protein (SEQ ID NO:340) within amino acids from about 1to about 109; from about 1 to about 54; from about 1 to about 44; fromabout 1 to about 34; from about 1 to about 24; and from about 1 to about14. In a second embodiment, the antibody of the invention binds one ormore of an epitope of the mature GDF8 protein (SEQ ID NO:340) withinamino acids from about 35 to about 109; from about 45 to about 109; fromabout 55 to about 109; from about 65 to about 109; from about 75 toabout 109; from about 85 to about 109; from about 92 to about 109; orfrom about 95 to about 109. In a third embodiment, the antibody orantigen-binding fragment of the antibody binds within an epitope of themature human GDF8 protein from about amino acid residue 48 to about 72;from about 48 to about 69; from about 48 to about 65; from about 52 toabout 72; from about 52 to about 65; or from about 56 to about 65.

In a related embodiment, the invention provides an antibody orantigen-binding fragment thereof that competes for specific binding toGDF8 with another antibody comprising aHCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 amino acid sequence combination ofSEQ ID NO:36/38/40/44/46/48, 116/118/120/124/126/128,228/230/232/236/238/240, 362/364/366/370/372/374, or378/380/382/386/388/390. In one embodiment, the antibody orantigen-binding fragment of the invention competes for specific bindingto GDF8 with another antibody comprising a HCVR/LCVR amino acid sequencepair of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106,114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234,242/250, 258/266, 274/282, 290/298, 306/314, 114/322, 360/368, or376/384. In yet another related embodiment, the invention provides anantibody or antigen-binding fragment thereof that recognizes the epitopeon GDF8 that is recognized by another antibody comprising a HCDRs/LCDRsamino acid sequence combination of SEQ ID NO: 36/38/40/44/46/48,116/118/120/124/126/128, 228/230/232/236/238/240,362/364/366/370/372/374, or 378/380/382/386/388/390. In one embodiment,the antibody or antigen-binding fragment of the invention recognizes theepitope on GDF8 that is recognized by another antibody comprising aHCVR/LCVR amino acid sequence pair of SEQ ID NO:2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298,306/314, 114/322, 360/368, or 376/384.

The present invention also features a composition comprising arecombinant human or humanized anti-human GDF8 antibody and anacceptable carrier. Further included in the invention are vectors andhost cells comprising vectors which contain nucleic acid moleculesencoding the human anti-GDF8 antibody of the invention, as well asmethods of producing these novel antibodies, comprising growing a hostcell comprising nucleic acid encoding the anti-GDF8 antibody of theinvention or an antibody fragment, under conditions permittingproduction of the protein and recovering the protein so produced.

The present invention also features methods for inhibiting GDF8 activityusing an antibody, or antigen-binding portion thereof, of the invention.In one embodiment, the method comprises administering an antibody orantibody fragment of the invention, to a human subject suffering from adisorder which is ameliorated by inhibition of GDF8 activity. Inpreferred embodiments, the human subject treated with the antibody orantibody fragment of the invention is in need of improving glucosehomeostasis, decreasing fat mass, increasing insulin sensitivity,improving kidney function and/or decreasing fat accumulation. Theantibody or antibody fragment of the invention is useful for treating,preventing or inhibiting a disease or condition characterized by boneloss, including osteoporosis, osteopenia, osteoarthritis and bonefractures, treating metabolic syndrome, counteracting muscle wastingfrom sustained administration of a glucocorticoid or a steroid hormoneor muscle loss related to muscle dystrophy, muscle atrophy, musclewasting syndrome, sarcopenia and cachexia.

Other objects and advantages will become apparent from a review of theensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Immunoblot of Limited Proteolysis of Human GDF8 with ProteinaseK. Gels were nonreducing 18% SDS-PAGE with 0.2 μg GDF8 loaded in eachlane, and 2 μg/ml of antibodies either control I (A), 1A2 (B) or 21-E9(C). Lane 1: digest time 10 min, 1 μg GDF8, 0 μg Proteinase K; Lane 2:digest time 10 min, GDF8 1 μg, 1 μg Proteinase K; Lane 3: digest time 10min, 1 μg GDF8, 6 μg Proteinase K; Lane 4: digest time 45 min, 1 μgGDF8, 0 μg Proteinase K; Lane 5: digest time 45 min, GDF8 1 μg, 1 μgProteinase K; Lane 6: digest time 45 min, 1 μg GDF8, 6 μg Proteinase K.

FIG. 2. Immunoblot of Limited Proteolysis of Human GDF8 with High Dosesof Proteinase K. Gels were nonreducing 18% SDS-PAGE with 0.2 μg GDF8loaded in each lane, and 2 μg/ml of either control I (A) or 1A2 (B).Lane 1: digest time 16 hr, 0 μg GDF8, 96 μg Proteinase K; Lane 2: digesttime 16 hr, GDF8 4 μg, 0 μg Proteinase K; Lane 3: digest time 16 hr, 4μg GDF8, 24 μg Proteinase K; Lane 4: digest time 16 hr, 4 μg GDF8, 96 μgProteinase K; Lane 5: digest time 1 hr, GDF8 4 μg, 24 μg Proteinase K;Lane 6: digest time 1 hr, 4 μg GDF8, 96 μg Proteinase K; Lane 7: digesttime 10 min, GDF8 0 μg, 0 μg Proteinase K; Lane 8: digest time 10 min, 4μg GDF8, 24 μg Proteinase K.

FIGS. 3A and 3B. Graphs illustrating the percent starting glucose levelsover time in mice subjected to an insulin tolerance test before (FIG.3A) and after (FIG. 3B) antibody treatment. FIGS. 4A and 4B. Alignmentof the amino acid sequences of the heavy chain CDRs (FIG. 4A) and lightchain CDRs (FIG. 4B) from exemplary anti-GDF8 antibodies H4H1657N2 andH4H1669P, illustrating the consensus sequences shared between thesesequences.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

“Human Growth Differentiation Factor-8”, “GDF8” and “myostatin” are usedinterchangeably to refer to the protein encoded by the nucleic acidsequence of SEQ ID NO:338 and the protein having the amino acid sequenceof SEQ ID NO:339 (propeptide) and 340 (mature protein).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the invention, the FRs of theanti-GDF8 antibody (or antigen-binding portion thereof) may be identicalto the human germline sequences, or may be naturally or artificiallymodified. An amino acid consensus sequence may be defined based on aside-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)₂ fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR)). Other engineered molecules,such as diabodies, triabodies, tetrabodies and minibodies, are alsoencompassed within the expression “antigen-binding fragment,” as usedherein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a VH domain associated with a V_(L)domain, the V_(H) and V_(L) domains may be situated relative to oneanother in any suitable arrangement. For example, the variable regionmay be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) or V_(L)-V_(L)dimers. Alternatively, the antigen-binding fragment of an antibody maycontain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (X) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (XII) V_(L)-C_(H)1-C_(H)2-C_(H)3; (Xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric VH or VLdomain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656(1998)).

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Specific binding can becharacterized by a dissociation constant of 1×10⁻⁶ M or less. Methodsfor determining whether two molecules specifically bind are well knownin the art and include, for example, equilibrium dialysis, surfaceplasmon resonance, and the like. For example, an antibody that“specifically binds” human GDF8, as used in the context of the presentinvention, includes antibodies that bind human GDF8 or portion thereof(e.g., a peptide comprising at least 6 contiguous amino acids of SEQ IDNO:340) with a K_(D) of less than about 1000 nM, less than about 500 nM,less than about 300 nM, less than about 200 nM, less than about 100 nM,less than about 90 nM, less than about 80 nM, less than about 70 nM,less than about 60 nM, less than about 50 nM, less than about 40 nM,less than about 30 nM, less than about 20 nM, less than about 10 nM,less than about 5 nM, less than about 4 nM, less than about 3 nM, lessthan about 2 nM, less than about 1 nM or less than about 0.5 nM, asmeasured in a surface plasmon resonance assay. (See, e.g., Example 3,herein). An isolated antibody that specifically binds human GDF8 may,however, have cross-reactivity to other antigens, such as GDF8 moleculesfrom other species.

The term “high affinity” antibody refers to those antibodies capable ofbinding to GDF8 with a dissociation constant (K_(D)) of about 10⁻⁸ M orless, about 10⁻⁹M or less, about 10⁻¹⁹ M or less, about 10⁻¹¹M or less,or about 10⁻¹² M or less, as measured by surface plasmon resonance,e.g., BIACORE™ or solution-affinity ELISA.

By the term “slow off rate” or “Koff” is meant an antibody thatdissociates from GDF8 with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁻⁴ s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

A “neutralizing” or “blocking” antibody, is intended to refer to anantibody whose binding to GDF8 results in inhibition of the biologicalactivity of GDF8. This inhibition of the biological activity of GDF8 canbe assessed by measuring one or more indicators of GDF8 biologicalactivity. These indicators of GDF8 biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art (see examples below).

The fully-human anti-GDF8 antibodies disclosed herein may comprise oneor more amino acid substitutions, insertions and/or deletions in theframework and/or CDR regions of the heavy and light chain variabledomains as compared to the corresponding germline sequences. Suchmutations can be readily ascertained by comparing the amino acidsequences disclosed herein to germline sequences available from, forexample, public antibody sequence databases. The present inventionincludes antibodies, and antigen-binding fragments thereof, which arederived from any of the amino acid sequences disclosed herein, whereinone or more amino acids within one or more framework and/or CDR regionsare back-mutated to the corresponding germline residue(s) or to aconservative amino acid substitution (natural or non-natural) of thecorresponding germline residue(s) (such sequence changes are referred toherein as “germline back-mutations”). A person of ordinary skill in theart, starting with the heavy and light chain variable region sequencesdisclosed herein, can easily produce numerous antibodies andantigen-binding fragments which comprise one or more individual germlineback-mutations or combinations thereof. In certain embodiments, all ofthe framework and/or CDR residues within the V_(H) and/or V_(L) domainsare mutated back to the germline sequence. In other embodiments, onlycertain residues are mutated back to the germline sequence, e.g., onlythe mutated residues found within the first 8 amino acids of FR1 orwithin the last 8 amino acids of FR4, or only the mutated residues foundwithin CDR1, CDR2 or CDR3. Furthermore, the antibodies of the presentinvention may contain any combination of two or more germlineback-mutations within the framework and/or CDR regions, i.e., whereincertain individual residues are mutated back to the germline sequencewhile certain other residues that differ from the germline sequence aremaintained. Once obtained, antibodies and antigen-binding fragments thatcontain one or more germline back-mutations can be easily tested for oneor more desired property such as, improved binding specificity,increased binding affinity, improved or enhanced antagonistic oragonistic biological properties (as the case may be), reducedimmunogenicity, etc. Antibodies and antigen-binding fragments obtainedin this general manner are encompassed within the present invention.

The present invention also includes anti-GDF8 antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-GDF8 antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein. In one embodiment, the antibody comprises anHCVR having an amino acid sequence selected from SEQ ID NO:360 and 376with 8 or fewer conservative amino acid substitutions. In anotherembodiment, the antibody comprises an HCVR having an amino acid sequenceselected from SEQ ID NO:360 and 376 with 6 or fewer conservative aminoacid substitutions. In another embodiment, the antibody comprises anHCVR having an amino acid sequence selected from SEQ ID NO:360 and 376with 4 or fewer conservative amino acid substitutions. In anotherembodiment, the antibody comprises an HCVR having an amino acid sequenceselected from SEQ ID NO:360 and 376 with 2 or fewer conservative aminoacid substitutions. In one embodiment, the antibody comprises an LCVRhaving an amino acid sequence selected from SEQ ID NO:368 and 384 with 8or fewer conservative amino acid substitutions. In another embodiment,the antibody comprises an LCVR having an amino acid sequence selectedfrom SEQ ID NO:368 and 384 with 6 or fewer conservative amino acidsubstitutions. In another embodiment, the antibody comprises an LCVRhaving an amino acid sequence selected from SEQ ID NO:368 and 384 with 4or fewer conservative amino acid substitutions. In another embodiment,the antibody comprises an LCVR having an amino acid sequence selectedfrom SEQ ID NO:368 and 384 with 2 or fewer conservative amino acidsubstitutions.

In certain embodiments, antibody or antibody fragment of the inventionmay be conjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin, a chemotherapeutic drug, and immunosuppressant or aradioisotope.

An “isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, tissueor cell in which the antibody naturally exists or is naturally producedis an “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell, as well as an antibody that has been subjected to at least onepurification or isolation step. According to certain embodiments, anisolated antibody may be substantially free of other cellular materialand/or chemicals.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. For example, an antibody is said tospecifically bind an antigen when the K_(D) is less than or equal to10⁻⁸ M, less than or equal to 10⁻⁹ M, or less than or equal to 10⁻¹⁰ M.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60, 70%, 80% or 90% w/w ofa protein sample, usually about 95%, and preferably over 99% pure.Protein purity or homogeneity may be indicated by a number of means wellknown in the art, such as polyacrylamide gel electrophoresis of aprotein sample, followed by visualizing a single polypeptide band uponstaining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The term “polypeptide analog or variant” as used herein refers to apolypeptide that is comprised of a segment of at least 25 amino acidsthat has substantial identity to a portion of an amino acid sequence andthat has at least one of the following properties: (1) specific bindingto GDF8 under suitable binding conditions, or (2) ability to block thebiological activity of GDF8. Typically, polypeptide analogs or variantscomprise a conservative amino acid substitution (or insertion ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, at least 50, 60, 70, 80, 90,100, 150 or 200 amino acids long or longer, and can often be as long asa full-length naturally-occurring polypeptide.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmutations of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton 1984 W. H. Freeman andCompany, New York; Introduction to Protein Structure (Branden & Tooze,eds., 1991, Garland Publishing, NY); and Thornton et at. 1991 Nature354:105, which are each incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (see, for example, Fauchere (1986) J. Adv. Drug Res.15:29; and Evans et al. (1987) J. Med. Chem. 30:1229, which areincorporated herein by reference. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may also be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo et al.(1992) Ann. Rev. Biochem. 61:387, incorporated herein by reference), forexample, by adding internal cysteine residues capable of formingintramolecular disulfide bridges which cyclize the peptide.

The term “percent sequence identity” in the context of nucleic acidsequences refers to the residues in two sequences which are the samewhen aligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides ormore, usually at least about 18 nucleotides, more usually at least about24 nucleotides, typically at least about 28 nucleotides, more typicallyat least about 32 nucleotides, and preferably at least about 36, 48 ormore nucleotides. There are a number of different algorithms known inthe art which can be used to measure nucleotide sequence identity. Forinstance, polynucleotide sequences can be compared using FASTA, Gap orBestfit, which are programs in Wisconsin Package Version 10.0, GeneticsComputer Group (GCG), Madison, Wis. FASTA, which includes, e.g., theprograms FASTA2 and FASTA3, provides alignments and percent sequenceidentity of the regions of the best overlap between the query and searchsequences (Pearson (1990) Methods Enzymol. 183:63-98 and (2000) MethodsMol. Biol. 132:185-219, each herein incorporated by reference). Unlessotherwise specified, default parameters for a particular program oralgorithm are used. For instance, percent sequence identity betweennucleic acid sequences can be determined using FASTA with its defaultparameters (a word size of 6 and the NOPAM factor for the scoringmatrix) or using Gap with its default parameters as provided in GCGVersion 6.1, herein incorporated by reference.

A reference to a nucleic acid sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid molecule havinga particular sequence should be understood to encompass itscomplementary strand, with its complementary sequence. Generally, theart uses the terms “percent sequence identity”, “percent sequencesimilarity” and “percent sequence homology” interchangeably. In thisapplication, these terms shall have the same meaning with respect tonucleic acid sequences.

The term “substantial similarity”, or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, indicates that,when optimally aligned with appropriate nucleotide insertions ordeletions with another nucleic acid (or its complementary strand), thereis nucleotide sequence identity in at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98% or atleast about 99% of the nucleotide bases, as measured by any well-knownalgorithm of sequence identity, such as FASTA, BLAST or Gap, asdiscussed above.

As applied to polypeptides, the term “substantial identity” or“substantially identical” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least about 80% sequence identity, at least about90%, at least about 95%, at least about 98% or at least about 99%sequence identity. Preferably, residue positions which are not identicaldiffer by conservative amino acid substitutions. A “conservative aminoacid substitution” is one in which an amino acid residue is substitutedby another amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of similarity may be adjustedupwards to correct for the conservative nature of the substitution.Means for making this adjustment are well-known to those of skill in theart. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, hereinincorporated by reference. Examples of groups of amino acids that haveside chains with similar chemical properties include 1) aliphatic sidechains: glycine, alanine, valine, leucine and isoleucine; 2)aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; and 6) sulfur-containing side chainsare cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256:1443-45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000), supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially blastp or tblastn, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389 402, each of which is hereinincorporated by reference.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, at least about 20 residues, atleast about 24 residues, at least about 28 residues, or at least about35 residues. When searching a database containing sequences from a largenumber of different organisms, it is preferable to compare amino acidsequences.

The term “effective amount” is a concentration or amount of an antibodyor antigen-binding fragment of an antibody which results in achieving aparticular stated purpose. An “effective amount” of an anti-GDF8antibody or antigen-binding fragment of an antibody thereof may bedetermined empirically. Furthermore, a “therapeutically effectiveamount” is a concentration or amount of an anti-GDF8 antibody orantigen-binding fragment thereof which is effective for achieving astated therapeutic effect. This amount may also be determinedempirically.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the present invention to make human antibodiesthat specifically bind to GDF8.

Using VELOCIMMUNE™ technology or any other known method for generatingmonoclonal antibodies, high affinity chimeric antibodies to GDF8 areinitially isolated having a human variable region and a mouse constantregion. As in the experimental section below, the antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc. The mouse constant regions arereplaced with a desired human constant region to generate the fullyhuman antibody of the invention, for example wild-type or modified IgG1or IgG4. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

In general, the antibodies of the instant invention possess very highaffinities, typically possessing K_(D) of from about 10⁻¹² through about10⁻⁹ M, when measured by binding to antigen either immobilized on solidphase or in solution phase. The mouse constant regions are replaced withdesired human constant regions to generate the fully human antibodies ofthe invention, for example wild-type IgG1 (SEQ ID NO:335) or IgG4 (SEQID NO:336), or modified IgG1 or IgG4 (for example, SEQ ID NO:337). Whilethe constant region selected may vary according to specific use, highaffinity antigen-binding and target specificity characteristics residein the variable region.

Bioequivalents

The anti-GDF8 antibodies and antibody fragments of the present inventionencompass proteins having amino acid sequences that vary from those ofthe described antibodies, but that retain the ability to bind humanGDF8. Such variant antibodies and antibody fragments comprise one ormore additions, deletions, or substitutions of amino acids when comparedto parent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, the anti-GDF8antibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-GDF8 antibody or antibody fragment that isessentially bioequivalent to an anti-GDF8 antibody or antibody fragmentof the invention. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-GDF8 antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-GDF8 antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Epitope Mapping and Related Technologies

To screen for antibodies which bind to a particular epitope (e.g., thosewhich block binding of IgE to its high affinity receptor), a routinecross-blocking assay such as that described in Harlow and Lane (1990)supra can be performed. Other methods include alanine scanning mutants,peptide blots (Reineke (2004) Methods Mol Biol 248:443-63) (hereinspecifically incorporated by reference in its entirety), or peptidecleavage analysis. In addition, methods such as epitope excision,epitope extraction and chemical modification of antigens can be employed(Tomer (2000) Protein Science 9:487-496) (herein specificallyincorporated by reference in its entirety).

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (US2004/0101920, herein specifically incorporated by reference in itsentirety). Each category may reflect a unique epitope either distinctlydifferent from or partially overlapping with epitope represented byanother category. This technology allows rapid filtering of geneticallyidentical antibodies, such that characterization can be focused ongenetically distinct antibodies. When applied to hybridoma screening,MAP may facilitate identification of rare hybridoma clones that producemAbs having the desired characteristics. MAP may be used to sort theanti-GDF8 antibodies of the invention into groups of antibodies bindingdifferent epitopes.

The invention includes anti-GDF8 antibodies and antigen-bindingfragments of antibodies which bind specific epitopes of human GDF8 (SEQID NO:340) and are capable of blocking the biological activity of GDF8.In one embodiment, the antibody or antigen-binding fragment thereofbinds within an epitope comprising amino acids residues 1 to 109; 1 to54; 1 to 44; 1 to 34; 1 to 24; and 1 to 14. In another embodiment, theantibody or antigen-binding fragment thereof binds within an epitopecomprising of amino acid residues 65 to 72; 35 to 109; 45 to 109; 55 to109; 65 to 109; 75 to 109; 85 to 109; 92 to 109; or 95 to 109. Inanother embodiment, the antibody or antigen-binding fragment thereofbinds within an epitope comprising amino acid residue 48 to 72; 48 to69; 48 to 65; 52 to 72; 52 to 65; or 56 to 65. In specific embodiments,the antibody or antigen-binding fragment thereof may bind within 2 ormore epitopes.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind wild-type mature GDF8 (SEQ ID NO:340) but donot bind isolated peptides having less than the full amino acid sequenceof SEQ ID NO:340. For example, the invention includes anti-GDF8antibodies that bind wild-type mature GDF8 (SEQ ID NO:340) but do notbind isolated peptides consisting of 10 to 40 contiguous amino acids ofSEQ ID NO:340. The invention also includes anti-GDF8 antibodies that donot bind any linear epitopes within wild-type mature GDF8. In certainembodiments of the present invention, the anti-GDF8 antibodies bindwild-type mature human GDF8 comprising SEQ ID NO:340 but do not bind oneor more isolated GDF8 peptides having an amino acid sequence selectedfrom the group consisting of amino acids 1-14, 1-18, 17-42, 48-65,48-69, 48-72, 52-65, 52-72, 56-65, 56-72, 65-72, 73-90, 75-105 and91-105, of SEQ ID NO:340. In certain embodiments, the anti-GDF8antibodies do not bind any of the aforementioned GDF8 peptides. Methodsfor determining whether a given antibody is able to bind a particularGDF8 peptide are known to persons of ordinary skill in the art. Oneexemplary method is illustrated by Example 7 herein, in which GDF8peptides are attached to microspheres, antibodies are added to thepeptide-conjugated microspheres, and, following washing steps,antibody-bound microspheres are detected. The absence of boundantibodies indicates that the antibodies do not bind the particularpeptides being tested.

The present invention also includes isolated human antibodies, orantigen-binding fragments thereof, that specifically bind to wild-typemature human GDF8 (e.g., a protein or polypeptide comprising SEQ IDNO:340), but do not bind to a chimeric GDF8 construct in which certainamino acids of GDF8 are replaced with the corresponding amino acidsequence(s) from a non-identical but related protein such as TGFβ-1. Inone example, the chimeric construct is a GDF8/TGFβ-1 chimera in whichamino acids 48-72 of mature GDF8 are replaced with the correspondingamino acid sequence of TGFβ-1 (e.g., amino acids 49-76 of TGFβ-1). Anexample of one such chimera is represented by SEQ ID NO:352 (seeExamples 4 and 6 herein). Thus, in certain embodiments, the antibodiesof the invention specifically bind to wild-type mature human GDF8 (SEQID NO:340) but do not bind to the chimeric GDF8/TGFβ-1 construct of SEQID NO:352, indicating that the epitope to which such antibodies bindincludes or encompasses amino acids located within residues 48 to 72 ofSEQ ID NO:340. Blocking bioassays, such as the assay set forth inExample 4 herein, can also be used to indirectly ascertain if anantibody binds wild-type mature human GDF8 (SEQ ID NO:340) and does notbind a chimeric GDF8/TGFβ-1 construct, e.g., the construct of SEQ IDNO:352. For example, an antibody which blocks the bioactivity ofwild-type mature human GDF8 but does not block the bioactivity of achimeric GDF8/TGFβ-1 is deemed to bind to the portion of GDF8 that isreplaced by the corresponding TGFβ-1 sequence in the chimeric construct.

Similarly, the present invention also includes isolated humanantibodies, or antigen-binding fragments thereof, that block wild-typemature GDF8-mediated activity in a bioassay but do not block activity ofa chimeric GDF8 construct (e.g., a GDF8/TGFβ-1 chimera in which aminoacids 48-72 of mature GDF8 are replaced with the corresponding aminoacid sequence of TGFβ-1 (e.g., SEQ ID NO:352)). An exemplary GDF8bioassay that can be used in the context of this aspect of the inventionis the GDF8-inducible luciferase assay set forth in Example 4 herein,although other similar bioassays capable of measuring the cellularactivity of GDF8 are contemplated herein as well.

The present invention includes anti-GDF8 antibodies that bind to thesame epitope as any of the specific exemplary antibodies describedherein. Likewise, the present invention also includes anti-GDF8antibodies that cross-compete for binding to GDF8 or a GDF8 fragmentwith any of the specific exemplary antibodies described herein.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-GDF8 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-GDF8antibody of the invention, the reference antibody is allowed to bind toa GDF8 protein or peptide under saturating conditions. Next, the abilityof a test antibody to bind to the GDF8 molecule is assessed. If the testantibody is able to bind to GDF8 following saturation binding with thereference anti-GDF8 antibody, it can be concluded that the test antibodybinds to a different epitope than the reference anti-GDF8 antibody. Onthe other hand, if the test antibody is not able to bind to the GDF8molecule following saturation binding with the reference anti-GDF8antibody, then the test antibody may bind to the same epitope as theepitope bound by the reference anti-GDF8 antibody of the invention.Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, Biacore, flow cytometryor any other quantitative or qualitative antibody-binding assayavailable in the art. In accordance with certain embodiments of thepresent invention, two antibodies bind to the same (or overlapping)epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antibodyinhibits binding of the other by at least 50% but preferably 75%, 90% oreven 99% as measured in a competitive binding assay (see, e.g., Junghanset al., Cancer Res. 1990: 50:1495-1502). Alternatively, two antibodiesare deemed to bind to the same epitope if essentially all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies aredeemed to have “overlapping epitopes” if only a subset of the amino acidmutations that reduce or eliminate binding of one antibody reduce oreliminate binding of the other.

To determine if an antibody competes for binding with a referenceanti-GDF8 antibody, the above-described binding methodology is performedin two orientations: In a first orientation, the reference antibody isallowed to bind to a GDF8 molecule under saturating conditions followedby assessment of binding of the test antibody to the GDF8 molecule. In asecond orientation, the test antibody is allowed to bind to a GDF8molecule under saturating conditions followed by assessment of bindingof the reference antibody to the GDF8 molecule. If, in bothorientations, only the first (saturating) antibody is capable of bindingto the GDF8 molecule, then it is concluded that the test antibody andthe reference antibody compete for binding to GDF8. As will beappreciated by a person of ordinary skill in the art, an antibody thatcompetes for binding with a reference antibody may not necessarily bindto the same epitope as the reference antibody, but may sterically blockbinding of the reference antibody by binding an overlapping or adjacentepitope.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, the anti-GDF8antibodies bind to human GDF8 but not to GDF8 from other species.Alternatively, the anti-GDF8 antibodies of the invention, in certainembodiments, bind to human GDF8 and to GDF8 from one or more non-humanspecies. For example, the anti-GDF8 antibodies of the invention may bindto human GDF8 and may bind or not bind, as the case may be, to one ormore of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit,goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus orchimpanzee GDF8.

Immunoconjugates

The invention encompasses a human or humanized anti-GDF8 monoclonalantibody conjugated to a therapeutic moiety (“immunoconjugate”), such asa cytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope. Cytotoxin agents include any agent that is detrimental tocells. Examples of suitable cytotoxin agents and chemotherapeutic agentsfor forming immunoconjugates are known in the art, see for example, WO05/103081, which is herein specifically incorporated by reference).

Multispecific Antibodies

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-GDF8 antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity. For example, the present invention includesbi-specific antibodies wherein one arm of an immunoglobulin is specificfor human GDF8 or a fragment thereof, and the other arm of theimmunoglobulin is specific for a second therapeutic target or isconjugated to a therapeutic moiety.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)CH3 domain and a second Ig CH3 domain, wherein the first and second IgCH3 domains differ from one another by at least one amino acid, andwherein at least one amino acid difference reduces binding of thebispecific antibody to Protein A as compared to a bi-specific antibodylacking the amino acid difference. In one embodiment, the first Ig CH3domain binds Protein A and the second Ig CH3 domain contains a mutationthat reduces or abolishes Protein A binding such as an H95R modification(by IMGT exon numbering; H435R by EU numbering). The second CH3 mayfurther comprise a Y96F modification (by IMGT; Y436F by EU). Furthermodifications that may be found within the second CH3 include: D16E,L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N,V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, andV82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 antibodies. Variations on the bi-specific antibody formatdescribed above are contemplated within the scope of the presentinvention.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theantibodies or antigen-binding fragments thereof of the presentinvention. The administration of therapeutic compositions in accordancewith the invention will be administered with suitable carriers,excipients, and other agents that are incorporated into formulations toprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-waterand water-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. Any of the foregoing mixtures may be appropriate intreatments and therapies in accordance with the present invention,provided that the active ingredient in the formulation is notinactivated by the formulation and the formulation is physiologicallycompatible and tolerable with the route of administration. See alsoPowell et al. “Compendium of excipients for parenteral formulations” PDA(1998) J Pharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to beadministered, target disease, conditions, route of administration, andthe like. When the antibody of the present invention is used fortreating various conditions and diseases associated with GDF8, forexample, muscular dystrophy, muscle atrophy, muscle wasting syndrome,sarcopenia and cachexia, in an adult patient, it is advantageous tointravenously administer the antibody of the present invention normallyat a single dose of about 0.01 to about 20 mg/kg body weight, about 0.1to about 10 mg/kg body weight, or about 0.1 to about 5 mg/kg bodyweight. Depending on the severity of the condition, the frequency andthe duration of the treatment can be adjusted. In other parenteraladministration and oral administration, the antibody can be administeredin a dose corresponding to the dose given above. When the condition isespecially severe, the dose may be increased according to the conditionup to the amount that causes significant side effects, if any.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533).

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system, for example, with the use of a pump orpolymeric materials. In another embodiment, a controlled release systemcan be placed in proximity of the composition's target, thus requiringonly a fraction of the systemic dose.

Examples of the composition for oral administration include solid orliquid dosage forms, specifically, tablets (including dragees andfilm-coated tablets), pills, granules, powdery preparations, capsules(including soft capsules), syrup, emulsions, suspensions, etc. Such acomposition is manufactured by publicly known methods and contains avehicle, a diluent or an excipient conventionally used in the field ofpharmaceutical preparations. Examples of the vehicle or excipient fortablets are lactose, starch, sucrose, magnesium stearate, and the like.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to 500 mg per dosage form in a unit dose;especially in the form of injection, it is preferred that the aforesaidantibody is contained in about 5 to 100 mg and in about 10 to 250 mg forthe other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the present invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with GDF8 activity. More specifically, the antibodies of thepresent invention are useful for the treatment of any condition oraffliction which can be improved by increasing muscle strength/powerand/or muscle mass and/or muscle function in an individual, or byfavorably altering metabolism (carbohydrate, lipid and proteinprocessing) by blocking GDF8 activity. Exemplary diseases, disorders andconditions that can be treated with the anti-GDF8 antibodies of thepresent invention include, but are not limited to, sarcopenia, cachexia(either idiopathic or secondary to other conditions, e.g., cancer,chronic renal failure, or chronic obstructive pulmonary disease), muscleinjury, muscle wasting and muscle atrophy, e.g., muscle atrophy orwasting caused by or associated with disuse, immobilization, bed rest,injury, medical treatment or surgical intervention (e.g., hip fracture,hip replacement, knee replacement, etc.) or by necessity of mechanicalventilation. The anti-GDF8 antibodies of the invention may also be usedto treat, prevent or ameliorate diseases such as cancer, obesity,diabetes, arthritis, multiple sclerosis, muscular dystrophy, amyotrophiclateral sclerosis, Parkinson's disease, osteoporosis, osteoarthritis,osteopenia, metabolic syndromes (including, but not limited to diabetes,obesity, nutritional disorders, organ atrophy, chronic obstructivepulmonary disease, and anorexia).

The present invention includes therapeutic administration regimens whichcomprise administering an anti-GDF8 antibody of the present invention incombination with at least one additional therapeutically activecomponent. Non-limiting examples of such additional therapeuticallyactive components include other GDF8 antagonists (e.g., small moleculeinhibitors of GDF8 or other GDF8 antibodies or binding molecules),growth factor inhibitors, immunosuppressants, anti-inflammatory agents,metabolic inhibitors, enzyme inhibitors, and cytotoxic/cytostaticagents. The additional therapeutically active component(s) may beadministered prior to, concurrent with, or after the administration ofthe anti-GDF8 antibody of the present invention.

Diagnostic Uses of the Antibodies

The anti-GDF8 antibodies of the present invention may also be used todetect and/or measure GDF8 in a sample, e.g., for diagnostic purposes.For example, an anti-GDF8 antibody, or fragment thereof, may be used todiagnose a condition or disease characterized by aberrant expression(e.g., over-expression, under-expression, lack of expression, etc.) ofGDF8. Exemplary diagnostic assays for GDF8 may comprise, e.g.,contacting a sample, obtained from a patient, with an anti-GDF8 antibodyof the invention, wherein the anti-GDF8 antibody is labeled with adetectable label or reporter molecule. Alternatively, an unlabeledanti-GDF8 antibody can be used in diagnostic applications in combinationwith a secondary antibody which is itself detectably labeled. Thedetectable label or reporter molecule can be a radioisotope, such as 3H,¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such asfluorescein isothiocyanate, or rhodamine; or an enzyme such as alkalinephosphatase, β-galactosidase, horseradish peroxidase, or luciferase.Specific exemplary assays that can be used to detect or measure GDF8 ina sample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in GDF8 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of GDF8 protein, orfragments thereof, under normal or pathological conditions. Generally,levels of GDF8 in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal GDF8 levels or activity) will be measured to initiallyestablish a baseline, or standard, level of GDF8. This baseline level ofGDF8 can then be compared against the levels of GDF8 measured in samplesobtained from individuals suspected of having a GDF8 related disease orcondition.

EXAMPLES Example 1 Generation of Human Antibodies to Human GDF8

Mice may be immunized by any method known in the art (see, for example,Harlow and Lane supra). In one embodiment, GDF8 antigen is administereddirectly, with an adjuvant to stimulate the immune response, to aVELOCIMMUNE® mouse comprising DNA encoding human Ig heavy and kappalight chain variable regions. Suitable adjuvants include complete andincomplete Freund's adjuvant, MPL+TDM adjuvant system (Sigma), or RIBI(muramyl dipeptides) (see O'Hagan 2000 Vaccine Adjuvant, by Human Press,Totawa, N.J.). The antibody immune response is monitored by standardantigen-specific immunoassay. When a desired immune response isachieved, in one embodiment, antibody-expressing B cells are harvestedand fused with mouse myeloma cells to preserve their viability and formhybridoma cell lines. The hybridoma cell lines are screened and selectedto identify cell lines that produce antigen-specific antibodies.

Alternatively, antigen-specific hybridoma cells may be isolated by flowcytometry. Briefly, after fusion to myeloma cells, pooled hybridomacells are grown for 10 days in HAT medium. The cells are then harvestedand stained with biotin-labeled GDF8 at 2 μg/ml for one hour, followedby addition of phycoerythrin-streptavidin. The fluorescence-labeledcells are sorted by flow cytometry (single cell per well into 96 wellplates containing hybridoma growth medium), cultured for 8-10 days, andconditioned media screened for the presence of functionally desirablemonoclonal antibodies.

In another embodiment, anti-GDF8 antibodies generated via directisolation of splenocytes. Antigen-specific antibodies are isolateddirectly from antigen-immunized B cells without fusion to myeloma cells,as described in U.S. 2007/0280945A1, herein specifically incorporated byreference in its entirety. Stable recombinant antibody-expressing CHOcell lines are established from the isolated proper recombinants.

Example 2 Gene Utilization Analysis

To analyze the structure of antibodies produced, the nucleic acidsencoding antibody variable regions were cloned and sequenced. From thenucleic acid sequence and predicted amino acid sequence of theantibodies, gene usage was identified for each antibody chain. Table 1sets forth the gene usage for selected antibodies in accordance with theinvention. Antibody identifier (HCVR/LCVR): 21-E5 (SEQ ID NO:34/42);21-B9 (SEQ ID NO:18/26); 21-E9 (SEQ ID NO:98/106); 21-A2 (SEQ IDNO:2/10); 22-D3 (SEQ ID NO:50/58); 22-E6 (SEQ ID NO:66/74); 22-G10 (SEQID NO:82/90); 1A2 (SEQ ID NO:226/234); 20B12 (SEQ ID NO:274/282); 58C8(SEQ ID NO:242/250); 19F2 (SEQ ID NO:258/266); 8D12-1 (SEQ IDNO:114/122); 4E3-7 (SEQ ID NO:194/202); 9B11-12 (SEQ ID NO:162/170); 4B9(SEQ ID NO:226/234); 1H4-5 (SEQ ID NO:210/218); 9B4-3 (SEQ IDNO:178/186); 3E2-1 (SEQ ID NO:290/298); 4G3-25 (SEQ ID NO:306/314);4B6-6 (SEQ ID NO:130/138); H4H1657N2 (SEQ ID NO:360/368); H4H1669P (SEQID NO:376/384).

TABLE 1 Heavy Chain Light Chain Variable Variable Region Region AntibodyVH D JH VK JK 21-E5 4-39 3-22 5 1-17 1 21-B9 4-39 3-22 5 1-17 1 21-E94-39 3-22 5 1-17 1 21-A2 3-23 1-7 4 3-15 4 22-D3 3-21 5-5 4 1-17 2 22-E64-39 3-22 5 1-17 1 22-G10 2-5 1-7 4 1-16 4 1A2 3-23 1-7 3 3-15 4 20B123-23 6-13 6 3-15 4 58C8 3-23 1-7 3 3-15 2 19F2 3-30 1-26 4 2-28 3 8D12-13-30 1-7 4 2-137*  5* 4E3-7 3-23 1-7 4 3-15 4 9B11-12 3-23 1-7 3 3-15 44B9 3-23 1-7 3 3-15 4 1H4-5 3-30 6-13 6 3-15 4 9B4-3 3-23 1-7 4 3-15 43E2-1 4-34 4-4 4 1-9 4 3A4-3 3-21 5-5 4 1-17 1 4G3-25 3-30 3-3 4 2-28 54B6-6 3-30 1-7 4 2-137*  5* H4H1657N2 3-23 2-21 6 1-27 4 H4H1669P 3-333-9 6 1-12 4

Control Constructs Used in the Following Examples

Various control constructs (anti-GDF8 antibodies and other GDF8antagonists) were included in the following experiments for comparativepurposes. The control constructs are designated as follows: Control I: ahuman anti-GDF8 antibody with heavy and light chain variable regionshaving the amino acid sequences of the corresponding domains of “Myo29”(i.e., SEQ ID NOs: 16 and 18) as set forth in U.S. Pat. No. 7,261,893;Control II: a human anti-GDF8 antibody with heavy and light chainvariable regions having the amino acid sequences of the correspondingdomains of “2_(—)112_K” (i.e., SEQ ID NOs: 118 and 120) as set forth inUS 2006/0263354; Control III: ActRIIB-Fc fusion construct having theamino acid sequence of SEQ ID NO:391; and Control IV: variant ActRIIB-Fcfusion construct, identical to Control III except that alanine atposition 64 of SEQ ID NO:391 [A64] is replaced with an arginine [R64].(Not all control constructs were used in every Example).

Example 3 Antigen Binding Affinity Determination

Equilibrium dissociation constants (K_(D) values) for antigen binding toselected antibodies were determined by surface kinetics using areal-time biosensor surface plasmon resonance assay (BIACORE™ 2000).Each selected antibody was captured on either a goat anti-mouse IgGpolyclonal antibody surface or a goat anti-hFc polyclonal antibody(Jackson Immuno Research Lab) surface created through direct chemicalcoupling to a BIACORE™ chip to form a captured antibody surface. HumanGDF8 homodimer, hGDF11 homodimer, or hGDF5 homodimer at 25 nM wasinjected over the captured antibody surfaces, and antigen-antibodybinding and dissociation were monitored in real time at roomtemperature. Kinetic analysis was performed to calculate K_(D),dissociation rate constants (kd), association rate constants (ka) andhalf-life of antigen/antibody complex dissociation (Table 2).

TABLE 2 GDF8 GDF11 Antibody K_(D) (nM) T_(1/2)(min) K_(D) (nM)T_(1/2)(min) 21-E5 0.26 138 0.12 116 21-B9 0.12 126 0.064 133 21-E9 0.14155 0.07 221 21-A2 0.40 78 0.90 21 22-D3 1.23 34 1.09 21 22-E6 0.26 1480.12 87 22-G10 0.250 71 0.73 50 1A2 0.32 60 0.30 28 20B12 0.86 39 2.08 258C8 0.62 56 0.44 30 19F2 0.50 38 — — 8D12-1 0.66 95 1.87 23 4E3-7 1.8927 1.33 29 9B11-12 1.45 39 1.41 29 4B9 0.55 55 1.09 34 1H4-5 0.95 541.48 24 9B4-3 1.18 50 1.08 32 3E2-1 2.55 45 0.70 79 3A4-3 1.07 137 0.5171 4G3-25 3.90 25 1.41 39 4B6-6 0.95 121 0.55 82 Control I 0.05 191 0.08136 Control II 0.3 41 — —

The foregoing experiment was also carried out with GDF8 applied over acaptured antibody surface of candidate antibodies H4H1669P or H4H1657N2.Preliminary data showed a very slow off rate for both antibodies,suggesting a K_(D) of 1-2 pM or less.

K_(D) values for antigen binding to selected antibodies were alsodetermined as described above with a modified running buffer that doesnot contain BSA.

TABLE 3 GDF8 GDF11 Antibody K_(D) (nM) T_(1/2)(min) K_(D) (nM)T_(1/2)(min) 1A2-mIgG 0.018 152 0.926 1 1A2-hIgG 0.006 340 0.640 6 8D120.016 840 NB Control I 0.002 1301 0.001 105 Control II 0.071 62 1.580 7

Additional antigen binding experiments were conducted in which GDF8 andGDF11 were applied over a surface of selected anti-GDF8 antibodies andcontrol antibodies at 25° C. and 37° C. Equilibrium dissociationconstants (K_(D) values) for antigen binding to selected antibodies weredetermined by surface kinetics using a real-time biosensor surfaceplasmon resonance assay (BIACORE™ T100). Each selected antibody orcontrol was captured on a goat anti-hFc polyclonal antibody (JacksonImmuno Research Lab Cat #109-005-098) surface created through directchemical coupling to a BIACORE™ CM5 sensor chip to form a capturedantibody surface. Various concentrations (2.5-0.625 nM, 2-folddilutions) of hGDF8 homodimer or hGDF11 homodimer (or in someexperiments, Activin A) were injected over the captured antibodysurfaces, and antigen-antibody binding and dissociation were monitoredin real time. Kinetic analysis was performed to calculate K_(D),dissociation rate constants (kd), association rate constants (ka) andhalf-life of antigen/antibody complex dissociation. Results aresummarized in Table 4. (NB=no binding observed).

TABLE 4 Antigen 25° C. 37° C. Inhibitor Tested K_(D) (M) T½ (min) K_(D)(M) T½ (min) H4H1669P GDF8 3.93E−11 131 5.84E−11 40 GDF11 NB NB NB NBH4H1657N2 GDF8 2.83E−11 202 3.79E−11 91 GDF11 NB NB NB NB Activin A NBNB not determined 1A2-hIgG1 GDF8 6.23E−11  96 4.62E−11 39 GDF11 NB NB NBNB Control I GDF8 1.03E−11 273 2.44E−11 70 (Myo29) GDF11 1.46E−11 2212.74E−11 56 Activin A NB NB not determined Control III GDF8 3.07E−11 1116.40E−12 104  (ActRIIB- GDF11 2.38E−11 132 9.92E−12 53 hFc [A64])Activin A 8.50E−12 196 not determined Control IV GDF8 2.13E−11 238 notdetermined (ActRIIB- GDF11 3.00E−12 231 hFc [R64]) Activin A 3.00E−12439 Isotype GDF8 NB NB NB NB negative GDF11 NB NB NB NB control ActivinA NB NB not determined antibody

As shown above, antibodies H4H1669P, H4H1657N2, and 1A2-hIgG1 of thepresent invention all exhibited strong binding to GDF8 but no binding toGDF11. By contrast, the control molecules showed binding to both GDF8and GDF11.

Example 4 Antibody Blocking of Smad2/Luciferase Response

GDF8-Inducible Luciferase Assay. A bioassay was developed to determinethe ability of selected anti-GDF8 antibodies to neutralize GDF8-mediatedor GDF11-mediated cellular function in vitro using an engineered A204cell line (human rhabdomyosarcoma cells, ATCC) that contains a GDF8 orGDF11-responsive promoter driving luciferase expression. Inhibition ofGDF8 or GDF11-inducible luciferase activity was determined as follows:Cells were seeded onto 96-well plate at 2×10⁴ cells/well in media andincubated overnight at 37° C., 5% CO₂. Antibody protein (in serialdilutions starting from 25 nM in cell media) was added to the wells ofA204/Smad2 cells in triplicate on two plates; GDF8 or GDF11 (0.8 nM) wasadded to each well. The plates were incubated at 37° C., 5% CO₂ for 6hours. Luciferase activity was determined by adding BRIGHT-GLO®Substrate (Promega) and IC₅₀ values determined (Table 5).

TABLE 5 IC₅₀ (nM) Antibody GDF8 GDF11 21-E5 8.50 35 21-B9 0.62 1.2 21-E90.99 1.2 21-A2 9.70 >10 22-D3 >20 >25 22-E6 2.20 22.4 22-G10 10.50 >251A2 0.80 1400 20B12 >20 >25 58C8 1.80 >25 19F2 >20 >25 8D12-1 2.40 >254E3-7 10.40 >25 9B11-12 5.50 >1000 4B9 0.47 >25 3A4-3 3.10 >204G3-25 >25 >25 Control I 0.62 0.94

The ability of selected anti-GDF8 antibodies to neutralize GDF8-mediatedor GDF11-mediated cellular function was further analyzed as describedabove with varied concentrations of GDF8 or GDF11. (Table 6). (nd=notdetermined; NB=no binding).

TABLE 6 IC₅₀ (nM) GDF8 GDF11 Antibody 0.5 nM 1 nM 0.4 nM 0.8 nM 1A20.196 0.363 ~600 ~800 8D12 3.11 5.55 >1000 >1000 21-E5 2.34 2.98 6.910.5 H4H1657N2 0.78 nd NB nd H4H1669P 0.90 nd NB nd Control I 0.1720.398 0.255 0.459 Control II 1.6 4.15 >1000 >1000

The bioassay described above was repeated using a GDF8/TGFβ-1 chimericconstruct as the activating peptide. In particular, a chimera consistingof mature GDF8 with amino acids 48-72 replaced with the correspondingamino acids of TGFβ-1 was used in this experiment (SEQ ID NO:352, alsoreferred to herein as “GDF8/TGFβ[48-72]”). This was produced from anexpression construct encoding the entire human GDF8 precursor with thehuman TGFβ-1 sequence replacing the corresponding GDF8 sequence.Bioactivity was assayed in conditioned medium produced by transienttransfection of the GDF8/TGFβ[48-72] construct in CHO cells. Expressionand processing were assessed by Western blot. The conditioned medium wasconcentrated 20-fold, heated to 80° C. for 5 minutes to inactivate thebound GDF8 propeptide, and assessed for activity in serial dilution inthe bioassay. While the precise concentration of chimeric protein wasnot determined in these experiments, the typical concentration was inthe range of 1-10 ug/ml prior to concentration. As described above,cells containing a GDF8-responsive promoter driving luciferaseexpression were seeded onto a 96-well plate. Selected antibodies (inserial dilutions starting at 100 nM in cell media) were added to anamount of the GDF8/TGFβ[48-72] chimeric protein conditioned mediumdetermined to give maximal response. This mixture was preincubated for45 minutes and added to the reporter cells. Luciferase activity wasmeasured and approximate IC₅₀ values were calculated, as shown in Table7 below. (NB=no blocking).

TABLE 7 Antibody Blocking of GDF8/TGFβ[48-72] Bioactivity Antibody IC₅₀(nM) 1A2-hFc 0.357 8D12-mFc NB H2M1657N2 NB H1H1669P NB Control I 0.402

The GDF8/TGFβ[48-72] chimeric construct was able to activate luciferaseexpression in this assay, and 1A2-hFc and Control I were able to blockbioactivity of this construct. However, antibodies H2M1657N2 andH1H1669P failed to block the bioactivity of the GDF8/TGFβ[48-72]chimeric construct. Since these two antibodies were shown to block thebioactivity of wild-type GDF8 in this assay system (see Table 6), it canbe concluded that H2M1657N2 and H1H1669P most likely exert theirbiological effects by interacting with an epitope within amino acids48-72 of GDF8.

Example 5 Immunoblotting hGDF8 Fragments Generated by Proteinase KDigestion

Western blot analysis was used to determine the immunoreactivity of testand control mAbs human GDF8 proteolytically digested with Proteinase K.Enzyme reactions containing 1 μg human GDF8 and 0, 1 or 6 μg ProteinaseK were incubated for either 10 or 45-min in digestion buffer. Equalaliquots containing 20% of the amount of GDF8 present in the reactionmixture (200 ng) was loaded into 3 separate 18% SDS-PAGE non-reducinggels and electroblotted to PVDF membranes. Each membrane was incubatedwith primary antibody at 2 μg/ml followed by the appropriate secondaryantibody conjugated to HRP. Shown in FIG. 1A-C, is the resultingimmunoreactivity detected for the control mAb I, 1A2 mAb and 21-E9 mAb,respectively. Results show a loss of GDF8 reactivity for the control mAb(A) in lanes 3 and 6. In marked contrast, antibody 1A2 retainsimmunoreactivity to a smaller GDF8 fragment of approximately 17-19 kD inmolecular weight (FIG. 1B).

The experiment was repeated with digestion times of up 10 min, 1 hr or16 hr, in the presence of 0 or 4 μg of GDF8, and 0, 24 or 96 μgProteinase K. The results (FIG. 2A-B) show that in the absence ofProteinase K, both control and 1A2 mAbs were immunoreactive with fulllength mature (undigested) GDF8 (see FIG. 2A-B, Lanes 2 and 7). In thepresence of Proteinase K, immunoreactivity is lost for the control I mAbat all time points. In marked contrast, 1A2 mAb remained immunoreactivewith digested human GDF8 fragment (FIG. 2B, Lanes 3-6 and 8). Theseresults indicate that 1A2 mAb is immunoreactive with a smaller fragmentof GDF8 that remains intact in the presence of Proteinase K, whereascontrol mAb loses immunoreactivity to the smaller fragment (17-19 kD).

A modified Western blot analysis was used to further determine the hGDF8epitope for selected anti-hGDF8 antibodies. The modification being thatbefore the hGDF8 specific primary antibody was incubated with themembrane, each anti-hGDF8 antibody was pre-incubated with 1000 fold or50 fold molar excess of hGDF8 peptide fragments of 1-14 amino acids,17-42 amino acids, 48-72 amino acids, or 75-105 amino acids. The resultsshow that pre-incubation of peptide fragment 48-72 amino acids at 50fold molar excess was able to block the binding of antibody 8D12 tohGDF8.

Example 6 Antibody Binding to GDF8 Chimeras

Twelve chimeric GDF8 pro-proteins were made. Table 8 shows the maturechimeric GDF8 protein structures. The chimeric GDF8 proteins comprisedtwo sets: one set having various substitutions of GDF8 sequences withBMP2 sequences, the other set having various substitutions of GDF8sequences with TGFβ1 sequences. These chimeric proteins were used totest and localize antibody binding.

TABLE 8 Sub- stituted Mature SEQ Chimera GDF8 ID Name Fragment MatureChimeric GDF8 Structure NO: B1  1-15 BMP2.1-13.GDF8.16-109 347 T1  1-15TGFb1.1-15.GDF8.16-109 348 B17 17-42 GDF8.1-16.BMP2.15-42.GDF8.43-109349 T17 17-42 GDF8.1-16,TGFβ1.17-43.GDF8.43-109 350 B48 48-72GDF8.1-47.BMP2.48-77.GDF8.73-109 351 T48 48-72GDF8.1-47.TGFβ1.49-76.GDF8.73-109 352 B65 65-72GDF8.1-64.BMP2.68-77GDF8.73-109 353 T65 65-72GDF8.1-64.TGFβ1.69-76.GDF8.73-109 354 B75  75-105GDF8.1-74.BMP2.80-110DGF8.106-109 355 T75  75-105GDF8.1-74.TGFβ1.79-108.GDF8.106-109 356 B91  91-105GDF8.1-90.BMP2.96-110.GDF8.106-109 357 T91  91-105GDF8.1-90.TGFβ1.95-10GDF8.106-109 358

The various chimeric GDF8 pro-proteins were transiently transfected inan engineered stable CHO.hFurin cell line. A similar Western Blotanalysis as described above was used to detect the binding of variousanti-hGDF8 antibodies to each of the chimeric GDF8. Briefly, 10 μg ofCHO supernatant were loaded onto each lane of an SDS-PAGE (non-reducingor reducing) gel and electroblotted onto a PVDF membrane. The membranewas then incubated with an anti-GDF8 antibody at 2 μg/ml followed byexposure to the appropriate secondary antibody conjugated to HRP. Asshown in Table 9, antibody 8D12 was not able to bind either B48 or T48.The result indicated that amino acids 48 to 72 of mature GDF8participate in the binding of antibody 8D12 to GDF8.

TABLE 9 Non Reducing Reducing GDF8 Protein Control I 8D12 Control II 1A24A7 8D2 Wild Type GDF8 + + + + + + B48 + − + + + − B65 + + + + + +B91 + + + + + + T48 + − + + + − T65 + + + + + +

Example 7 Antibody Binding to hGDF8 Peptides

Fourteen peptides (Table 10) were generated from mature hGDF8 (SEQ IDNO:340). Unmodified peptides, N-terminal biotinylated peptides (N-term),or C-terminal biotinylated peptides (C-term) were used to test andlocalize antibody binding. Full-length hGDF8, hGDF11 and unmodifiedpeptides were each individually amine-coupled to xMAP® Multi-AnalyteCOOH Microspheres (or beads). Each of the biotinylated peptides wasbound to xMAP® Multi-Analyte LumAvidin Microspheres. Peptide-bound beadssuspension were then mixed with an equal volume of blocking buffer (PBS,1% BSA, 0.05% Tween20, 0.05% Sodium azide) and then distributed into a96 well filter plate (Millipore, MULTISCREEN® BV). Control and testanti-hGDF8 antibodies, at 2.5 μg/ml were then added to the peptide-boundbeads suspension and were allowed to bind to the beads at RT, overnight.The antibody-incubated beads were then washed twice with PBST (PBS+0.05%Tween20) and incubated with either Phycoerythrin (PE) conjugatedanti-hFC or PE-conjugated anti-mFC antibodies at RT for 45 min. Thebeads were washed again and the antibody binding signal to variouspeptides were detected with either LUMINEX® 100™ or 200™ instruments. Asshown in Table 10, anti-hGDF8 antibody 8D12 is able to bind to peptides4, 5, 6, 7, 8, and 9. By contrast, anti-hGDF8 antibody H4H1657N2 did notbind any of the peptides (data not shown).

TABLE 10 Control Control 21- No. Peptide Modification I II E5 1A2 8D12 1 1-14 Unmodified − − − − − N-term − − − − − C-term − − − − − 2  1-18Unmodified − − − − − N-term − − − − − C-term − ++ − +++ +/− 3 17-42Unmodified − − − − − N-term − − − − − C-term − − − − − 4 48-65Unmodified − − − − +++ C-term − − − − +++++ 5 48-69 Unmodified − − − −+++ C-term − − − − +++++ 6 48-72 Unmodified − + + + +++ N-term − − − − +C-term − − − − +++++ 7 52-65 Unmodified − − − − +++ C-term − − − − +++++8 52-72 Unmodified − − − − ++ C-term − − − − +++++ 9 56-65 C-term − − −− ++ 10 56-72 Unmodified − − − − − C-term − − − − + 11 65-72 Unmodified− − − − − N-term − − − − − C-term − − − − − 12 73-90 Unmodified − − − −+/− N-term − +/− − + +/− C-term + − − − + 13  75-105 Unmodified − − − −− N-term − − − − − C-term − − − − − 14  91-105 Unmodified − − − − −N-term − − − − − C-term − − − − −

Example 8 Effect of Human Anti-GDF8 Antibodies on the Binding GDF8 toActivin RIIB

Mature hGDF8 was first amine-coupled to Luminex® beads. The hGDF8-coatedLuminex® beads were then incubated with various anti-hGDF8 antibodies,at 1.25 μg/ml, for 2 hr at room temperature. Human Activin RIIB-mFc wasthen added to the bead-antibody mixture and incubated for an additional2 hr at room temperature. The beads were then washed and stained withR-phycoerythrin (R-PE)-conjugated anti-mFc polyclonal antibody and meanfluorescence intensity (MFI) was measured. As shown in Table 11,although Control mAb I and antibody 21-E5 were both able to block thebinding between hGDF8 and hActivin RIIB, Control mAb II was only able topartially block binding. Antibody 1A2 was not able to block the bindingof hGDF8 to its receptor, Activin RIIB (Table 11, n=3).

TABLE 11 Antibody MFI Negative Control 4,560 Control mAb I 58 ControlmAb II 1,653 1A2-hIgG 4,037 21-E5-hIgG 275

Example 9 Antibody 8D12 Variants

Antibody 8D12 variants with modified LCVR were generated by modifyingone or more of the following amino acids of the LCVR of 8D12: A7S or T,A8P, 9PL, S18P, V19A, M21I, K27Q, F41Y, V42L, R44K, R55L or T, M56G orL, N58Y, L59R, A75D, R79K, A105G, L109V, and L111I.

The binding affinity (K_(D)) of the antibody variants with respect tohGDF8 was determined using a real-time biosensor surface plasmaresonance assay (BIAcore™3000) described above in modified runningbuffer that does not contain BSA (Table 12).

TABLE 12 Antibody K_(D) (nM) T_(1/2) (min) 8D12 0.071 139 8D12-v2 0.52025 8D12-v3 0.380 46

The binding between antibody variants and hGDF8 peptides was also testedas described above in Example 7. Antibody variants 8D12-v2 and 8D12-v3showed strong binding to C-terminal biotinylated peptides 4, 5, 7, and10.

Example 10 Effect of Anti-GDF8 Antibodies on Skeletal Muscle Mass

The efficacy of selected anti-GDF8 mAbs for inducing skeletal musclehypertrophy was determined in vivo. Briefly, 20 male CB17 SCID mice,approximately 9 weeks old, were divided evenly according to body weightinto 4 groups. A selected mAb (Control I, 1A2, 21-E5, 8D12, 1A2-hIgG, orControl II) was injected at three increasing doses of 2.5 mg/kg/dose, 5mg/kg/dose, and 10 mg/kg/dose. The Fc fragment of human IgG was used asnegative control. Antibodies were administered intraperitoneally twicefor the first week and once a week for the following three weeks. On day28, mice were euthanized and weighed, and the tibialis anterior (TA)muscles, the gastrocnemius (GA) muscles, and quadriceps (Quad) muscles,heart, spleen, and kidney, were dissected and weighed. Tissues werenormalized to starting body weight, and percent change in weight overthe negative control was calculated. Six separate experiments wererepeated with antibodies: Control I, 1A2, 21-E5, 8D12, 1A2-hIgG, andControl II (Table 13-18). In addition, the experiment was also repeatedwith higher increasing doses of control I antibody at 10 mg/kg/dose, 30mg/kg/dose, and 50 mg/kg/dose (Table 19). Results are expressed aspercent increase over negative control±standard deviation.

TABLE 13 Negative Control I Control Dose 2.5 mg/kg 5 mg/kg 10 mg/kg 10mg/kg Body Weight  7.66 ± 1.37 9.92 ± 1.82 13.99 ± 1.21 0.00 ± 1.41 TAMuscle 13.84 ± 2.23 16.99 ± 2.16  13.68 ± 0.96 0.00 ± 2.71 GA Muscle10.58 ± 1.67 11.31 ± 2.44  12.90 ± 3.0  0.00 ± 3.36 Quad Muscle 14.79 ±1.55 15.99 ± 2.72  20.84 ± 2.09 0.00 ± 1.84 Heart  2.05 ± 3.04 2.96 ±2.96  7.55 ± 1.61 0.00 ± 2.45 Kidney  3.53 ± 1.39 3.56 ± 3.51  5.66 ±4.59 0.00 ± 2.82 Spleen 39.82 ± 6.78 45.26 ± 19.10  9.02 ± 3.08 0.00 ±5.47

TABLE 14 Negative 1A2 Control Dose 2.5 mg/kg 5 mg/kg 10 mg/kg 10 mg/kgBody Weight  6.08 ± 1.08  9.96 ± 1.32  9.92 ± 1.09 0.00 ± 1.76 TA Muscle15.56 ± 1.54 18.24 ± 4.49 20.69 ± 3.13 0.00 ± 4.47 GA Muscle 20.49 ±1.84 21.36 ± 2.79 24.36 ± 3.46 0.00 ± 3.59 Quad Muscle 26.92 ± 3.0730.15 ± 3.56 33.09 ± 4.46 0.00 ± 4.05 Heart  3.70 ± 1.31  6.37 ± 2.2712.42 ± 2.70 0.00 ± 4.10 Kidney  1.28 ± 2.89  2.89 ± 3.30  5.31 ± 3.290.00 ± 4.39 Spleen −8.07 ± 5.75 −10.00 ± 4.68   9.68 ± 9.19 0.00 ± 6.84

TABLE 15 Negative 21-E5 Control Dose 2.5 mg/kg 5 mg/kg 10 mg/kg 10 mg/kgBody 4.16 ± 0.87  4.14 ± 2.82 5.21 ± 1.58 0.00 ± 1.15 Weight TA 13.86 ±1.66  14.01 ± 2.41 10.32 ± 2.54  0.00 ± 1.65 Muscle GA 7.70 ± 1.86 12.94± 1.10 8.13 ± 2.36 0.00 ± 1.41 Muscle Quad 8.64 ± 1.31 13.57 ± 1.79 8.46± 3.22 0.00 ± 1.94 Muscle Heart −7.11 ± 1.00  −7.14 ± 3.17 −5.51 ± 1.58 0.00 ± 2.23 Kidney −6.8 ± 2.83  −3.2 ± 3.57 −0.32 ± 2.07  0.00 ± 3.93Spleen 29.81 ± 9.83  49.76 ± 7.86 10.85 ± 6.63  0.00 ± 5.76

TABLE 16 Negative 8D12 Antibody Control Dose 2.5 mg/kg 5 mg/kg 10 mg/kg10 mg/kg Body Weight 10.06 ± 0.86 12.76 ± 1.01 11.41 ± 1.35 0.00 ± 0.66TA Muscle 17.99 ± 1.53 21.30 ± 2.06 22.11 ± 3.20 0.00 ± 1.85 GA Muscle21.14 ± 1.19 23.10 ± 0.99 23.40 ± 3.72 0.00 ± 1.44 Quad Muscle 26.74 ±1.53 31.00 ± 1.61 28.80 ± 2.72 0.00 ± 0.94 Heart −1.61 ± 2.06  3.63 ±1.93  3.42 ± 2.52 0.00 ± 1.08 Kidney −1.06 ± 2.02 −4.26 ± 2.25 −5.52 ±3.83 0.00 ± 3.66 Spleen  4.33 ± 6.34 −9.04 ± 2.64 −1.85 ± 6.26 0.00 ±4.25

TABLE 17 1A2-hIgG Antibody Negative Control Dose 2.5 mg/kg 5 mg/kg 10mg/kg 10 mg/kg Body Weight 6.73 ± 1.51  3.84 ± 2.32  7.45 ± 2.91 0.00 ±0.76 TA Muscle 15.62 ± 2.4  12.09 ± 1.81 10.91 ± 3.33 0.00 ± 2.80 GAMuscle 16.29 ± 1.02  15.20 ± 2.54 15.67 ± 3.60 0.00 ± 2.13 Quad Muscle19.39 ± 2.92  20.03 ± 2.54 19.73 ± 3.83 0.00 ± 1.98 Heart 5.91 ± 3.81 5.39 ± 2.77  3.52 ± 3.16 0.00 ± 3.14 Kidney 1.70 ± 4.01  1.20 ± 1.73 1.39 ± 3.70 0.00 ± 3.27 Spleen −15.44 ± 5.6    −30.08 ± 6.63  −23.90 ±2.36   0.00 ± 11.32

TABLE 18 Control II Negative Control Dose 2.5 mg/kg 5 mg/kg 5 mg/kg BodyWeight 10.19 ± 1.20 10.83 ± 1.58 0.00 ± 1.17 TA Muscle 14.70 ± 3.2813.44 ± 4.13 0.00 ± 3.87 GA Muscle 19.44 ± 2.08 14.35 ± 3.36 0.00 ± 1.80Quad Muscle 18.92 ± 4.86 13.00 ± 2.18 0.00 ± 2.03 Heart  2.78 ± 5.09 2.71 ± 6.72 0.00 ± 0.69 Kidney  1.84 ± 2.65  0.41 ± 3.04 0.00 ± 2.12Spleen 12.32 ± 5.60  8.41 ± 3.38 0.00 ± 4.31

TABLE 19 Negative Control I Control Dose 10 mg/kg 30 mg/kg 50 mg/kg 10mg/kg Body Weight 9.20 ± 0.73 13.62 ± 1.38 12.55 ± 1.71 0.00 ± 1.18 TAMuscle 18.16 ± 1.51  25.58 ± 3.14 23.03 ± 2.00 0.00 ± 3.81 GA Muscle16.91 ± 2.08  23.72 ± 3.14 24.68 ± 2.73 0.00 ± 3.58 Quad Muscle 20.77 ±1.01  26.54 ± 3.68 25.67 ± 3.32 0.00 ± 4.63 Heart 1.55 ± 3.07  3.11 ±2.58  2.11 ± 2.23 0.00 ± 1.96 Kidney 3.09 ± 1.90 10.38 ± 5.08  8.89 ±3.59 0.00 ± 1.35 Spleen 8.22 ± 5.80  6.90 ± 5.87 −0.74 ± 1.85 0.00 ±7.38

A similar experiment was carried out using antibodies H4H1657N2 andH4H1669P and controls administered to SCID mice. In particular, maleSCID mice at 10 weeks of age were administered antibody subcutaneouslyat 10 mg/kg according to the following dosing schedule: 2× on week 1 and1×/week on weeks 2 and 3. The total treatment time was 28 days. For thisexperiment, 5 mice were administered an isotype negative controlantibody; 5 mice were administered Control I (Myo29); 6 mice wereadministered H4H1657N2; and 6 mice were administered H4H1669P. Resultsare summarized in Table 20 and are expressed as percent increase overnegative control±standard deviation.

TABLE 20 Isotype Control Control I H4H1657N2 H4H1669P Dose 10 mg/kg 10mg/kg 10 mg/kg 10 mg/kg Body Weight 0.00 ± 1.97 14.52 ± 2.68 10.28 ±0.95 10.70 ± 1.26 TA Muscle 0.00 ± 4.10 17.47 ± 3.09 25.53 ± 3.96 13.77± 2.01 GA Muscle 0.00 ± 1.97 19.46 ± 2.92 21.69 ± 1.67 13.39 ± 1.30 QuadMuscle 0.00 ± 3.59 14.05 ± 3.03 22.15 ± 3.47  9.87 ± 2.55 Heart 0.00 ±2.49  9.42 ± 1.63  5.40 ± 2.28 12.70 ± 2.67 White 0.00 ± 9.72 25.14 ±19  −8.05 ± 7.53  4.22 ± 6.19 Adipose Tissue

A similar experiment was also carried out using antibodies H4H1657N2 andH4H1669P and controls administered to C57 mice. In particular, male C57mice at 10 weeks of age were administered antibody subcutaneously at 10mg/kg according to the following dosing schedule: 2× per week for twoweeks. For this experiment, 5 mice were administered an isotype negativecontrol antibody; 5 mice were administered Control I (Myo29); 5 micewere administered H4H1657N2; and 6 mice were administered H4H1669P.Results are summarized in Table 21 and are expressed as percent increaseover negative control±standard deviation.

TABLE 21 Isotype Control Control I H4H1657N2 H4H1669P Dose 10 mg/kg 10mg/kg 10 mg/kg 10 mg/kg Body 0.00 ± 0.92 4.64 ± 0.99  5.11 ± 0.73 2.75 ±1.07 Weight TA 0.00 ± 4.26 8.92 ± 1.56 14.92 ± 3.09 7.62 ± 2.90 MuscleGA 0.00 ± 2.39 6.28 ± 2.10 14.20 ± 1.77 6.07 ± 3.80 Muscle Quad 0.00 ±2.46 3.53 ± 2.11 12.85 ± 2.69 1.24 ± 1.74 Muscle Heart 0.00 ± 2.28 −3.18± 2.70   1.39 ± 2.83 2.45 ± 3.95 White 0.00 ± 8.20 19.05 ± 7.72  −5.23 ±8.34 −4.53 ± 10.28 Adipose Tissue

Next, dose response experiments were carried out using antibodiesH4H1657N2 and H4H1669P in SCID mice. In particular, male SCID mice at 10weeks of age were administered control antibodies subcutaneously at 30mg/kg, and H4H1657N2 or H4H1669P at 2.5, 10 or 30 mg/kg, according tothe following dosing schedule: 2× per week on week 1 and 1× per weekthereafter. Results are summarized in Tables 22 and 23 and are expressedas percent increase over negative control±standard deviation.

TABLE 22 Isotype Control Control I H4H1657N2 Dose 30 mg/kg 30 mg/kg 2.5mg/kg 10 mg/kg 30 mg/kg Number of Mice (n) 5 5 5 5 5 Body Weight 0.00 ±1.38  7.46 ± 2.44 10.16 ± 0.92  6.51 ± 0.91 10.12 ± 1.68 TA Muscle 0.00± 2.55 16.13 ± 5.62 22.22 ± 2.75 19.79 ± 1.19 24.10 ± 3.62 GA Muscle0.00 ± 2.66 13.50 ± 5.20 21.94 ± 3.18 21.84 ± 2.94 25.01 ± 4.15 QuadMuscle 0.00 ± 2.06 16.89 ± 4.11 26.30 ± 3.72 26.28 ± 2.39 30.81 ± 2.98Heart 0.00 ± 2.67  3.23 ± 3.29 14.46 ± 1.55  2.70 ± 1.51  4.06 ± 2.53White Adipose Tissue  0.00 ± 13.91  5.08 ± 9.02 −23.70 ± 10.29 −16.77 ±7.54  −15.44 ± 11.43

TABLE 23 Isotype Control Control I H4H1669P Dose 30 mg/kg 30 mg/kg 2.5mg/kg 10 mg/kg 30 mg/kg Number of Mice (n) 5 5 5 5 5 Body Weight 0.00 ±1.47 13.77 ± 1.99 2.87 ± 1.66  5.72 ± 0.56 4.88 ± 1.11 TA Muscle 0.00 ±2.38 25.79 ± 2.37 7.82 ± 2.25 12.38 ± 2.56 11.86 ± 2.59  GA Muscle 0.00± 2.12 22.82 ± 1.25 2.68 ± 2.06 12.46 ± 1.30 10.55 ± 3.33  Quad Muscle0.00 ± 3.08 25.69 ± 3.65 4.10 ± 2.32 12.27 ± 1.63 9.43 ± 2.67 Heart 0.00± 0.91  7.78 ± 1.62 −0.01 ± 3.31   5.37 ± 1.71 2.84 ± 4.73 White AdiposeTissue 0.00 ± 9.41 12.60 ± 8.08 9.00 ± 4.12 −8.30 ± 3.71 −1.23 ± 7.03 

In a separate experiment, 5 groups of 6 male SCID mice at 10 weeks ofage were administered an isotype negative control antibodysubcutaneously at 10 mg/kg, and H4H1657N2 at 0.1, 0.75, 2.5, or 10mg/kg, according to the following dosing schedule: 2× per week on week 1and 1× per week thereafter for a total 28 days of treatment. Results aresummarized in Table 24, expressed as percent increase over negativecontrol±standard deviation.

TABLE 24 Isotype Control H4H1657N2 Dose 10 mg/kg 0.1 mg/kg 0.75 mg/kg2.5 mg/kg 10 mg/kg Body Weight 0.00 ± 0.55 4.24 ± 0.42 2.84 ± 1.03  6.87± 1.21  7.23 ± 1.66 TA Muscle 0.00 ± 3.89 3.10 ± 2.06 4.20 ± 3.78 18.63± 2.52 11.54 ± 2.15 GA Muscle 0.00 ± 1.65 2.75 ± 0.82 0.71 ± 3.15 15.86± 2.11 18.28 ± 2.84 Quad Muscle 0.00 ± 3.00 0.84 ± 1.69 −0.62 ± 1.75 15.01 ± 1.37 16.87 ± 2.85 Heart 0.00 ± 2.24 6.51 ± 1.53 1.35 ± 2.85 3.25 ± 1.21  3.08 ± 2.66 White Adipose  0.00 ± 13.65 −7.75 ± 8.36 −7.81 ± 10.76 −13.53 ± 8.00  −28.33 ± 4.51  Tissue

As shown in this Example, antibodies H4H1657N2 and H4H1669P producedsignificant increases in muscle mass when administered to mice over arange of doses.

Example 11 Effect of Anti-GDF8 Antibodies on Glucose Homeostasis

Anti-GDF8 antibodies of the invention were tested for their effects onglucose homeostasis and insulin sensitivity in a diet-induced-obesity(DIO) mouse model. In this experiment, DIO mice were obtained by feedingC57BL6 mice a high-fat diet (45% kcal fat) for 7 weeks starting at 9weeks of age. Starting at week 8, antibodies were administered at 30mg/kg twice a week for two more weeks, and the study was terminated aweek later (21 days post-treatment). The antibodies used in thisexperiment were H4H1669P and H4H1657N2, as well as an isotype negativecontrol antibody and Control I (anti-GDF8 antibody corresponding toMyo29). Insulin-tolerance tests were performed before and after antibodytreatment. Insulin (2 IU/kg) was administered by intraperitonealinjection following a 4 hour fast and glucose levels were measured. Theresults are illustrated in FIGS. 3A (prior to antibody treatment) and 3B(after antibody treatment).

As demonstrated in this experiment, myostatin inhibition byadministration of an anti-GDF8 antibody improved glucose homeostasis inDIO mice. A significant difference in glucose lowering in response to aninsulin bolus was noted between the anti-GDF8 antibody groups (H4H1669P,H4H1657N2 or Control I), and the isotype control antibody group.

Example 12 In Vivo Blockade of Muscle Atrophy by Administration ofH4H1657N2

C57 mice were used to determine the in vivo properties of H4H1657N2 inpreventing muscle atrophy induced by casting/immobilization anddexamethasone administration.

In the casting example, three groups of 8 C57 mice were anesthetized,and the ankle joint was immobilized at a 90° angle with casting materialfor 14 days. A fourth group was left unperturbed and used as anon-immobilized group control. During the 14 days of immobilization,mice were administered with an isotype negative control antibody;Control I (Myo29); or H4H1657N2. The three groups were injectedsubcutaneously with antibody at 30 mg/kg, 2× per week for two weeks,starting at the time of the immobilization. Results are summarized inTable 25, expressed as percent change over negative control±standarddeviation. The results showed that after 14 days of immobilization, thegroup that received treatment with H4H1657N2 antibody showed asignificant reduction in skeletal muscle loss versus the isotype controlgroup.

TABLE 25 (Muscle Atrophy Induced by Immobilization) Non- ImmobilizedImmobilized Isotype Isotype Control Control Control I H4H1657N2 TA 0.00± 1.74 −32.97 ± 4.98 −23.36 ± 5.80 −13.77 ± 4.23 Muscle GA 0.00 ± 1.85−24.34 ± 4.95  −7.64 ± 0.97  3.91 ± 6.02 Muscle

In the dexamethasone example, two groups of 5 C57 mice were anesthetizedand implanted with osmotic pumps subcutaneously that delivered 1μg/g/day of dexamethasone. Two additional groups were implanted withosmotic pumps that delivered saline and were used as controls. Duringthe 14 days of treatment, two groups of mice (one saline and onedexamethasone) were administered an isotype negative control antibody;and two groups of mice (one saline and one dexamethasone) wereadministered the H4H1657N2 antibody. Antibodies were injectedsubcutaneously at 30 mg/kg, 2× per week for two weeks. Antibodytreatment started at the time of the pump implantation. Results aresummarized in Table 26, expressed as percent change over negativecontrol±standard deviation. Comparison of the dexamethasone group thatreceived the H4H1657N2 antibody versus the group that received theisotype control indicates that treatment with the H4H1657N2 antibodyprevents the loss of muscle weights induced by dexamethasone treatment.

TABLE 26 (Muscle Atrophy Induced by Dexamethasone) Saline TreatedDexamethasone Treated Isotype Isotype Control H4H1657N2 ControlH4H1657N2 TA Muscle 0.00 ± 2.73 14.63 ± 1.35 −17.78 ± 2.11  3.25 ± 2.09GA Muscle 0.00 ± 3.02 20.73 ± 1.38 −18.97 ± 2.11 −5.08 ± 2.07 Quad 0.00± 3.91 20.46 ± 2.40 −23.94 ± 2.68 −4.26 ± 2.34 Muscle

Example 13 Specificity of H4H1657N2 In Vivo

To examine the specificity of H4H1657N2 in vivo, C57BL6 mice wereinjected with drug, and serum from treated mice was subjected to a massspectrometry based ligand “fishing” experiment. Briefly, drug(H4H1657N2, Control IV or isotype negative control) was injectedmultiple times (Day 0, D3, D7, D10) over a 14-day period into C57BL6mice. Animals were sacrificed on D14 and serum was incubated with antihuman Fc beads. The beads were washed and bound material was eluted withSDS-PAGE loading buffer. Eluted material was subjected to SDS-PAGE andgel slices corresponding to a molecular weight range of 5-20 kDa wereexcised. Samples were processed for mass spectrometry using standardreduction, alkylation and trypsinization conditions. Digests wereseparated on a nano C18 column and automatically spotted onto BrukerAnchor Targets. MALDI-MS (MS/MS) analysis (Bruker Ultraflextreme) wasperformed in an automated fashion, using LC-WARP (Bruker Daltonics). Themass spectra were searched using MASCOT (Matrix Science) and resultswere evaluated relative to the isotype control. The eluted proteins arelisted in Table 27.

TABLE 27 Antibody Administered: H4H1657N2 Control IV Protein(s) Eluted:GDF8 GDF8 GDF11 Inhibin beta A chain Inhibin beta B chain Inhibin beta Cchain

As shown in Table 27, only mouse GDF8 was identified as a bindingpartner for H4H1657N2 by this experiment. (The sequence of mouse andhuman GDF8 are identical.) By contrast, Control IV bound several othermembers of the TGF beta ligand family besides GDF8, including, interalia, GDF11. This experiment confirms the specificity of H4H1657N2 forGDF8 in an in vivo context.

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. An isolated human antibody or antigen-bindingfragment thereof that competes for binding to wild-type mature humanGDF8 (SEQ ID NO:340) with a reference antibody comprising: (a)HCDR1/HCDR2/HCDR3 amino acid sequences selected from the groupconsisting of SEQ ID NOs: 116/118/120; 362/364/366; and 376/380/382; and(b) LCDR1/LCDR2/LCDR3 amino acid sequences selected from the groupconsisting of SEQ ID NOs: 124/126/128; 370/372/374; and 386/388/390,wherein the isolated human antibody or antigen-binding fragment (a) doesnot bind to, or block the biological activity of a chimeric GDF8/TGFβ1construct having amino acids 48-72 of mature human GDF8 replaced withthe corresponding amino acid sequence of TGFβ1, and (b) does not bind toan isolated polypeptide consisting of amino acids 48-72 of SEQ ID NO:340.
 2. The isolated antibody or antigen-binding fragment of claim 1,wherein the chimeric GDF8/TGFβ1 construct comprises the amino acidsequence of SEQ ID NO:
 352. 3. The isolated antibody or antigen-bindingfragment of claim 1, wherein the antibody or antigen-binding fragmentcomprises heavy and light chain CDR amino acid sequences selected fromthe group consisting of: SEQ ID NOs: 116/118/120/124/126/128;362/364/366/370/372/374; and 376/380/382/386/388/390.
 4. The isolatedhuman antibody or antigen-binding fragment of claim 1, wherein theantibody or antigen-binding fragment has a heavy and light chainselected from the group consisting of: (a) an antibody comprising aheavy chain variable region (HCVR) having the amino acid sequence of SEQID NO:114, and a light chain variable region (LCVR) having the aminoacid sequence of SEQ ID NO:122; (b) an antibody comprising a HCVR havingthe amino acid sequence of SEQ ID NO:360, and a LCVR having the aminoacid sequence of SEQ ID NO:368; and (c) an antibody comprising a HCVRhaving the amino acid sequence of SEQ ID NO:376, and a LCVR having theamino acid sequence of SEQ ID NO:384.
 5. The isolated antibody orantigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment does not bind isolated GDF8 peptides havingamino acid sequences of amino acids 1-14, 1-18, 17-42, 48-65, 48-69,52-65, 52-72, 56-65, 56-72, 65-72, 73-90, 75-105 and 91-105, of SEQ IDNO:340.
 6. The isolated human antibody or antigen-binding fragment ofclaim 1, wherein the antibody or antigen-binding fragment thereof bindsto the same epitope on wild-type mature human GDF8 (SEQ ID NO:340) asthe reference antibody.
 7. The isolated antibody or antigen-bindingfragment of claim 1, wherein the antibody or antigen-binding fragmentcomprises HDCR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acidsequences, wherein: (a) the HCDR1 comprises an amino acid sequence ofthe formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO: 329), wherein X¹ is Gly;X² is Phe; X³ is Thr; X⁴ is Phe; X⁵ is Ser; X⁶ is Ala or Ser; X⁷ is Pheor Tyr; X⁸ is Gly or Ala; (b) the HCDR2 comprises an amino acid sequenceof the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO: 330), wherein X¹ isIle; X² Gly or Ser; X³ is Tyr or Gly; X⁴ Ser or Asp; X⁵ is Gly; X⁶ isGly; X⁷ is Ser or Asn; and X⁸ is Ala or Glu; (c) the HCDR3 comprises anamino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴ (SEQ ID NO:331), whereinX¹ is Ser or Ala; X² is Thr or Lys; X³ is Asp or Ile; X⁴ is Gly or Ser;X⁵ is Ala or His; X⁶ is Trp or Tyr; X⁷ is Lys or Asp; X⁸ is Met or Ile;X⁹ is Ser or Leu; X¹° is Gly or Ser; X¹¹ is Leu or Gly; X¹² is Asp orMet; X¹³ is Val or Asp; X¹⁴ is Val or absent; (d) the LCDR1 comprises anamino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶ (SEQ ID NO:332),wherein X¹ is Gln; X² is Asp or Gly; X³ is Ile; X⁴ is Ser; X⁵ is Asp orAsn; and X⁶ is Tyr or Trp; (e) the LCDR2 comprises an amino acidsequence of the formula X¹-X²-X³ (SEQ ID NO:333), wherein X¹ is Thr orAla; X² is Thr or Ala; and X³ is Ser; and (f) the LCDR3 comprises anamino acid sequence, of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ IDNO:334), wherein X¹ is Gln; X² is Lys or Gln; X³ is Ala or Tyr; X⁴ isAsp or Asn; X⁵ is Ser; X⁶ is Ala or Phe; X⁷ is Pro; X⁸ is Leu; and X⁹ isThr.
 8. A pharmaceutical composition comprising an antibody orantigen-binding fragment and a pharmaceutically acceptable carrier ordiluent; wherein the isolated human antibody or antigen-binding fragmentthereof competes for binding to wild-type mature human GDF8 (SEQ IDNO:340) with a reference antibody comprising (a) HCDR1/HCDR2/HCDR3 aminoacid sequences selected from the group consisting of SEQ ID NOs:116/118/120; 362/364/366; and 376/380/382; and (b) LCDR1/LCDR2/LCDR3amino acid sequences selected from the group consisting of SEQ ID NOs:124/126/128; 370/372/374; and 386/388/390, wherein the isolated humanantibody or antigen-binding fragment (a) does not bind to, or block thebiological activity of a chimeric GDF8/TGFβ1 construct having aminoacids 48-72 of mature human GDF8 replaced with the corresponding aminoacid sequence of TGFβ1, and (b) does not bind to an isolated polypeptideconsisting of amino acids 48-72 of SEQ ID NO:
 340. 9. The pharmaceuticalcomposition of claim 8, wherein the chimeric GDF8/TGFβ1 constructcomprises the amino acid sequence of SEQ ID NO:
 352. 10. Thepharmaceutical composition of claim 8, wherein the antibody orantigen-binding fragment has a heavy and light chain selected from thegroup consisting of: (a) an antibody comprising a heavy chain variableregion (HCVR) having the amino acid sequence of SEQ ID NO:114, and alight chain variable region (LCVR) having the amino acid sequence of SEQID NO:122; (b) an antibody comprising a HCVR having the amino acidsequence of SEQ ID NO:360, and a LCVR having the amino acid sequence ofSEQ ID NO:368; and (c) an antibody comprising a HCVR having the aminoacid sequence of SEQ ID NO:376, and a LCVR having the amino acidsequence of SEQ ID NO:384.
 11. The pharmaceutical composition of claim8, wherein the antibody or antigen-binding fragment comprises heavy andlight chain CDR amino acid sequences selected from the group consistingof: SEQ ID NOs: 116/118/120/124/126/128; 362/364/366/370/372/374; and376/380/382/386/388/390.
 12. The pharmaceutical composition of claim 8,wherein the antibody or antigen-binding fragment does not bind isolatedGDF8 peptides having amino acid sequences of amino acids 1-14, 1-18,17-42, 48-65, 48-69, 52-65, 52-72, 56-65, 56-72, 65-72, 73-90, 75-105and 91-105, of SEQ ID NO:340.
 13. The pharmaceutical composition ofclaim 8, wherein the antibody or antigen-binding fragment thereof bindsto the same epitope on wild-type mature human GDF8 (SEQ ID NO:340) asthe reference antibody.
 14. A therapeutic method for inhibiting GDF8activity in a patient, said method comprising administering to a patientin need thereof a human antibody or antigen-binding fragment thereof,wherein the isolated human antibody or antigen-binding fragment thereofcompetes for binding to wild-type mature human GDF8 (SEQ ID NO:340) witha reference antibody that comprises (a) HCDR1/HCDR2/HCDR3 amino acidsequences selected from the group consisting of SEQ ID NOs: 116/118/120;362/364/366; and 376/380/382; and (b) LCDR1/LCDR2/LCDR3 amino acidsequences selected from the group consisting of SEQ ID NOs: 124/126/128;370/372/374; and 386/388/390, wherein the isolated human antibody orantigen-binding fragment (a) does not bind to, or block the biologicalactivity of a chimeric GDF8/TGFβ1 construct having amino acids 48-72 ofmature human GDF8 replaced with the corresponding amino acid sequence ofTGFβ1, and (b) does not bind to an isolated polypeptide consisting ofamino acids 48-72 of SEQ ID NO:
 340. 15. The therapeutic method forinhibiting GDF8 activity in a patient of claim 14, wherein the chimericGDF8/TGFβ1 construct comprises the amino acid sequence of SEQ ID NO:352.
 16. The method of claim 14, wherein the patient is afflicted with,diagnosed with, or at risk of being afflicted with a disease or disorderselected from the group consisting of sarcopenia, cachexia, and muscleatrophy due to disuse or immobilization.
 17. The therapeutic method forinhibiting GDF8 activity in a patient of claim 14, wherein the antibodyor antigen-binding fragment has a heavy and light chain selected fromthe group consisting of: (a) an antibody comprising a heavy chainvariable region (HCVR) having the amino acid sequence of SEQ ID NO:114,and a light chain variable region (LCVR) having the amino acid sequenceof SEQ ID NO:122; (b) an antibody comprising a HCVR having the aminoacid sequence of SEQ ID NO:360, and a LCVR having the amino acidsequence of SEQ ID NO:368; and (c) an antibody comprising a HCVR havingthe amino acid sequence of SEQ ID NO:376, and a LCVR having the aminoacid sequence of SEQ ID NO:384.
 18. The therapeutic method forinhibiting GDF8 activity in a patient of claim 14, wherein the antibodyor antigen-binding fragment comprises heavy and light chain CDR aminoacid sequences selected from the group consisting of: SEQ ID NOs:116/118/120/124/126/128; 362/364/366/370/372/374; and376/380/382/386/388/390.
 19. The therapeutic method for inhibiting GDF8activity in a patient of claim 14, wherein the antibody orantigen-binding fragment does not bind isolated GDF8 peptides havingamino acid sequences of amino acids 1-14, 1-18, 17-42, 48-65, 48-69,52-65, 52-72, 56-65, 56-72, 65-72, 73-90, 75-105 and 91-105, of SEQ IDNO:340.
 20. The therapeutic method for inhibiting GDF8 activity in apatient of claim 14, wherein the antibody or antigen-binding fragmentthereof binds to the same epitope on wild-type mature human GDF8 (SEQ IDNO:340) as the reference antibody.